Recombinant Polypepties for Membrane Fusion and Uses Thereof

ABSTRACT

Disclosed is a recombinant polypeptide for facilitating membrane fusion. The recombinant polypeptide having a sequence with at least 80% sequence identity with the ectodomain of p14 fusion-associated small transmembrane (FAST) protein and having a functional myristoylation motif, a transmembrane domain from a FAST protein and a sequence with at least 80% sequence identity with the endodomain of p15 FAST protein. A targeting ligand can be added to the recombinant polypeptide for selective fusion. The recombinant polypeptide can be included in the membrane of a liposome, or the like, to facilitate the delivery of bioactive compounds, such as siRNA, or the recombinant polypeptide can be mixed with a lipid carrier and added to cultured cells to induce cell-cell fusion and heterokaryon formation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/877,101, filed Jun. 5, 2014 (issued Mar. 12, 2019 as U.S.Pat. No. 10,227,386; Atty. Dkt. 25000.49), which was a U.S.national-stage of PCT International Patent Application No.PCT/CA2011/001088 (nationalized), filed Sep. 29, 2011, which claims thebenefit of U.S. Provisional Patent Application No. 61/471,445, filedApr. 4, 2011 (expired), U.S. Provisional Patent Application No.61/438,155, filed Jan. 31, 2011 (expired), and U.S. Provisional PatentApplication No. 61/387,726, filed Sep. 29, 2010 (expired); the contentsof each of which is specifically incorporated herein in its entirety byexpress reference thereto.

FIELD OF THE INVENTION

The invention generally relates to a recombinant protein for membranefusion. More specifically, the invention relates to recombinant proteinhaving sequences related to fusion-associated small transmembraneproteins.

BACKGROUND OF THE INVENTION

Membrane fusion reactions are common in eukaryotic cells. Membranes arefused intracellularly in processes including endocytosis, organelleformation, inter-organelle traffic, and constitutive and regulatedexocytosis. Intercellularly, membrane fusion occurs during sperm-eggfusion and myoblast fusion.

Membrane fusion has been induced artificially by the use of liposomes,in which the cell membrane is fused with the liposomal membrane, and byvarious chemicals or lipids, which induce cell-cell fusion to produceheterokaryons. Naturally occurring proteins shown to induce fusion ofbiological membranes are mainly fusion proteins of enveloped viruses.

In liposome-based delivery systems, liposomes are used to encapsulatebioactive molecules inside lipid vesicles for delivery into the cell.There has been interest in using such delivery systems in treatingvarious cancer, since, in theory, the use of liposomes will allow for amore targeted approach to treating the cancer cells [Arias et al., Curr.Drug Targets, March 28 (2011)]. However, the polar lipid head groupsoriented on both surfaces of the lipid bilayer, along with an associatedwater layer, make spontaneous membrane fusion thermodynamicallyunfavorable. Moreover, the generalized release of the encapsulatedbioactive molecules to both the cancerous cells and the healthy cells ina cancer inflicted tissue makes traditional liposome-based deliverysystems less than perfect.

Various chemicals or lipids have been used to promote membrane fusion.However, these reagents usually exhibit cytotoxic effects [see, forexample, Iwanoto et al., in Biol. Pharm. Bull. 19:860-863 (1996) andMizugucji et al., in Biochem. Biophys. Res. Commun., 218:402-407(1996)].

It is generally believed that membrane fusion under physiologicalconditions is protein-mediated. This has lead to the development ofliposomes that contain fusion-promoting proteins (proteoliposomes), withdecreased cytotoxicity [see, for example, Cheng, Hum. Gene Ther.7:275-282 (1996); Hara et al., Gene, 159:167-174 (1995); and Findeis etal., Trends Biotechnol., 11:202-205 (1993)].

One particularly interesting group of proteins recently identified asfusion-promoting proteins are the fusion-associated small transmembrane(FAST) proteins. The FAST proteins are a unique family of membranefusion proteins encoded by the fusogenic retroviruses (Duncan et al.,Virology, 319:131-140 (2004). Currently, the FAST proteins include: p10,p14, p15 and p22. At 95 to 198 amino acids in size, the FAST proteinsare the smallest known viral membrane fusion proteins. Rather thanmediating virus-cell fusion, the FAST proteins are non-structural viralproteins that are expressed on the surfaces of virus-infected or-transfected cells, where they induce cell-cell fusion and the formationof multinucleated syncytia. A purified FAST protein, when reconstitutedinto liposome membranes, induces liposome-cell and liposome-liposomefusion, indicating the FAST proteins are bona fide membrane fusionproteins (Top et al., EMBO J. 24:2980-2988, 2005).

In contrast to most enveloped viral fusion proteins in which thecytoplasmic tail is extremely short relative to the overall size of theprotein, the FAST proteins all have an unusual topology that partitionsthe majority of the protein to the membrane and cytoplasm, exposingectodomains of just 20 to 43 residues to the extracellular milieu(Corcoran and Duncan, J. Viral., 78(8):4342-51, 2004; Dawe et al., J.Viral., 79(10):6216-26, 2005). Despite the diminutive size of theirectodomains, both p14 and p10 encode patches of hydrophobicity (HP)hypothesized to induce lipid mixing analogously to the fusion peptidesencoded by enveloped viral fusion proteins (Corcoran et al., J Biol Chem279(49): 51386-94, 2004; Shmulevitz et al., J Virol 78(6):2808-18,2004). The p14 HP is comprised of the N-terminal 21 residues of theprotein, but peptides corresponding to this sequence require theinclusion of the N-terminal myristate moiety to mediate lipid mixing.Nuclear magnetic resonance (NMR) spectroscopy revealed that two prolineresidues within the p14 HP form a protruding loop structure presentingvaline and phenylalanine residues at the apex and connected to the restof the protein by a flexible linker region (Corcoran et al., J Biol Chem279(49): 51386-94, 2004). The p10 HP on the other hand, flanked by twocysteine residues that form an intramolecular disulfide bond, may havemore in common with the internal fusion peptides of the Ebola virus andavian leukosis and sarcoma virus (ALSV) glycoproteins (Delos et al., JVirol., 74(4):1686-93, 2000; Delos and White, J. Virol., 74(20):9738-41,2000; Gallaher, 1996; Ito et al., J Virol., 73(10:8907-12, 1999;Ruiz-Arguello et al., J. Virol., 72(3):1775-81, 1998), and likely adoptsa cystine-noose structure that forces solvent exposure of conservedvaline and phenylalanine residues for membrane interactions (Barry etal., J. Biol. Chem., 285:16424, 2010). In contrast to p14 and p10, the20 residue ectodomain of p15 completely lacks a hydrophobic sequencethat could function as a traditional fusion peptide (Dawe et al., JVirol 79(10): 6216-26, 2005). In the absence of such a motif, the p15ectodomain instead encodes a polyproline helix that has been proposed tofunction as a membrane destabilizing motif.

There is a need in the art for the targeted delivery of bioactivemolecules encapsulated by liposomes.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a recombinantprotein that can induce membrane fusion.

According to an aspect of the present invention there is provided arecombinant polypeptide for facilitating membrane fusion, saidrecombinant polypeptide comprising: an ectodomain comprising a sequencewith at least 80% sequence identity with the sequence defined by SEQ IDNO:2 and comprising a functional myristoylation motif; a transmembranedomain comprising 23 amino acid residues, at least two hydrophobic,β-branched residues adjacent the ectodomain, three consecutive serineresidues immediately adjacent the at least two hydrophobic, β-branchedresidues, and a glycine residue at positions 7 and 13 from the junctionbetween the ectodomain and the first hydrophobic, β-branched residue;and an endodomain comprising a sequence with at least 80% sequenceidentity with the sequence defined by SEQ ID NO:3 or SEQ ID NO:4.

In one embodiment, the at least two hydrophobic, β-branched residues areselected from the group consisting of isoleucine and valine.

In another embodiment, the transmembrane domain is defined by a sequencehaving at least 80% sequence identity with the sequence defined by SEQID NO:11.

In a further embodiment, the ectodomain is defined by the sequencedefined by SEQ ID NO:2.

In a yet further embodiment, the endodomain is defined by the sequencedefined by SEQ ID NO:3 or SEQ ID NO:4.

According to another aspect of the present invention, there is provideda polypeptide defined by a sequence with at least 80% sequence identitywith the sequence defined by SEQ ID NO:1.

In one embodiment, the polypeptide is defined by the sequence defined bySEQ ID NO:1.

According to an aspect of the present invention, there is provided aliposome comprising the recombinant polypeptide as described above.

According to a further aspect of the present invention, there isprovided nucleic acid molecules encoding the recombinant polypeptides,and components thereof, described above.

According to a further aspect of the present invention, there isprovided expression vectors comprising the nucleic acid moleculesdescribed above.

According to another aspect of the present invention, there is provideda polypeptide comprising 23 amino acid residues, at least two N-terminalhydrophobic, β-branched residues, three consecutive serine residuesimmediately adjacent the at least two hydrophobic, β-branched residues,and a glycine residue at positions 7 and 13 from the N-terminus of thepolypeptide.

In one embodiment, the at least two hydrophobic, β-branched residues areselected from the group consisting of isoleucine and valine.

In another embodiment, the transmembrane domain is defined by a sequencehaving at least 80% sequence identity with the sequence defined by SEQID NO: 11.

According to another aspect of the present invention there is provided aliposome comprising a recombinant peptide embedded within the membraneof the liposome. The recombinant peptide comprises a fusion-associatedsmall transmembrane protein linked to a targeting ligand.

In one embodiment, the fusion-associated small transmembrane protein isselected from the family Reoviridae.

In another embodiment, the fusion-associated small transmembrane proteinis selected from the genus Orthoreovirus and Aquareovirus.

In a further embodiment, the genus Orthoreovirus comprises avian,mammalian and reptilian reoviruses.

In a yet further embodiment, the fusion-associated small transmembraneprotein is selected from the group consisting of p10, p14, p15 and p22.

In a still further embodiment, the fusion-associated small transmembraneis a chimera of two or more domains of p10, p14, p15 and p22.

In a further embodiment, the fusion-associated small transmembraneprotein is the recombinant polypeptide described above.

In another embodiment, the fusion-associated small transmembrane proteinis defined by a sequence with at least 80% sequence identity with thesequence defined by SEQ ID NO: 1.

In an additional embodiment, the targeting ligand is bombesin.

Furthermore, the recombinant peptide is defined by the sequence depictedin SEQ ID NO:17.

According to another aspect of the invention, there is provided arecombinant polypeptide comprising a fusion-associated smalltransmembrane protein linked to a targeting ligand.

In one embodiment, the fusion-associated small transmembrane protein isselected from the family Reoviridae.

In another embodiment, the fusion-associated small transmembrane proteinis selected from the genus Orthoreovirus and Aquareovirus.

In a further embodiment, the genus Orthoreovirus comprises avian,mammalian and reptilian reoviruses.

In a yet further embodiment, the fusion-associated small transmembraneprotein is selected from the group consisting of p10, p14, p15 and p22.

In a still further embodiment, the fusion-associated small transmembraneis a chimera of two or more domains of p10, p14, p15 and p22.

In a further embodiment, the fusion-associated small transmembraneprotein is the recombinant polypeptide described above.

In another embodiment, the fusion-associated small transmembrane proteinis defined by a sequence with at least 80% sequence identity with thesequence defined by SEQ ID NO:1.

In an additional embodiment, the targeting ligand is bombesin.

Furthermore, the recombinant peptide is defined by the sequence depictedin SEQ ID NO:17.

According to a further aspect of the invention, there is provided apolynucleotide encoding the recombinant polypeptide as described above.

According to a yet further aspect of the invention, there is provided ahost cell comprising the polynucleotide as described above.

According to an aspect of the present invention there is provided amethod for delivering siRNA to a cell. The method comprising exposing acell to a liposome comprising a membrane encircling an siRNA moleculeand a fusogenic protein spanning the membrane of the liposome. Thefusogenic protein being selected from p10, p14, 15, p22, the recombinantpolypeptide described above, and combinations or variations thereof.Preferably, the fusogenic protein is the recombinant polypeptidedescribed above, with or without the targeting ligand described above.

According to another aspect of the present invention there is provided amethod for producing a liposome comprising a membrane encircling ansiRNA molecule and a fusogenic protein spanning the membrane of theliposome. The method comprising the steps of: mixing siRNA with aliposome formulation to generate a core; encapsulating the core with alipid to generate a liposome; saturating the liposome with a detergent;mixing a detergent-suspended fusogenic protein with the detergentsaturated liposome in the presence of about 0.7-1.3% wt./vol. N-octylβ-D-glucopyranoside. The fusogenic protein being selected from p10, p14,p15, p22, the recombinant polypeptide described above, and combinationsor variations thereof. The detergent is then removed from the fusogenicprotein and liposome mixture; and the liposome comprising membraneencircling an siRNA molecule and a fusogenic protein spanning themembrane of the liposome isolated.

According to a further aspect of the present invention there is provideda liposome comprising an siRNA molecule encapsulated by a membraneembedded with a fusogenic protein. The fusogenic protein being selectedfrom the group consisting of p10, p14, p15, p22, the recombinantpolypeptide described above, and combinations and variations thereof.Preferably, the fusogenic protein is the recombinant polypeptidedescribed above, with or without the targeting ligand described above.

According to a yet further aspect of the present invention there isprovided use of the liposome described above for delivery of siRNA to acell.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription and accompanying drawings wherein:

(FIG. 1A) is a representation of a linear amino acid sequence of apolypeptide of the present invention (SEQ ID NO:1); (FIG. 1B) is arepresentation of a linear amino acid sequence of a p14 ectodomain (SEQID NO:2); (FIG. 1C) is a linear amino acid sequence of the polypeptidesequence of a p15 endodomain (SEQ ID NO:3 and SEQ ID NO:4); (FIG. 1D) isa linear polynucleotide sequence encoding a polypeptide of the presentinvention (SEQ ID NO:5); (FIG. 1E) is a linear polynucleotide sequenceencoding a p14 ectodomain (SEQ ID NO:6); and (FIG. 1F) is a linearpolynucleotide sequence encoding a p15 endodomain (SEQ ID NO:7);

FIG. 2 is a schematic representation of wild type p15 and p14, p14truncation (c78) and re-extension (c78p15) mutants, and p14end15,wherein “ecto” stands for ectodomain; “TM” stands for transmembranedomain; “endo” stands for endodomain, “HP” stands for hydrophobic patchand “+++” stands for polybasic region;

FIG. 3 is a graphical representation of QM5 cells transfected with p14,c78, c78p15 or p14end15, and the average number of syncytial nuclei perfield as determined from Giemsa stained monolayers 8 hourspost-transfection. Results are expressed as the means±standard deviationof a representative experiment done in triplicate;

FIG. 4A, FIG. 4B, and FIG. 4C is a graphical representation showing(FIG. 4A) the extent of pore formation as determined by co-transfectionof eGFP with empty pcDNA3 vector (not shown), p14, c78, c78e15 andp14end15 at a 1:14 ratio. Representative dot plots (left) and histograms(right) are shown from the same experiment, done in triplicate; (FIG.4B) percentage pore formation by dot plot or Overton subtraction fromthe experiment presented in panel (FIG. 4A). Results are presented asthe means±standard deviation of a representative experiment done threetimes in triplicate; (FIG. 4C) the extent of pore formation induced byp14 and p14end15 was determined as above except that a titration wascarried out and the indicated amounts of plasmid were transfected intoQM5 cells. The x-axis values represent the amount of p14 or p14end15plasmid transfected per 1 μg of total DNA;

FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D are graphical representationsshowing (FIG. 5A) the locations of binding of the anti-p14ecto antibodyto the ectodomains of both p14 and p14end15; (FIG. 5B) QM5 cellstransfected with p14 or p14end15 and anti-p14ecto antiserum (1:20) wasadded 2 hours post-transfection. Arrows indicate the borders ofsyncytia; (FIG. 5C) surface expression determined by transfecting QM5cells with p14, c78 or c78p15; (FIG. 5D) QM5 cells co-tranfected withthe indicated amounts of plasmid expressing p14G2A or p14end15G2A andpcDNA3, to a total of 1 μg, then stained and analysed by flow cytometry.Results are expressed as means±standard deviation of a representativeexperiment done in triplicate;

FIG. 6A, FIG. 6B, and FIG. 6C are graphical representations showing(FIG. 6A) a linear representation of p14 or a p14end15 harbouring a V9Tsubstitution. “TM” stands for transmembrane domain; “endo” stands forendodomain, “HP” stands for hydrophobic patch and “+++” stands forpolybasic region; (FIG. 6B) Giesma stains of QM5 cells transfected withp14V9T (a) or endV9T (b) for 24 hours. Arrows indicate the borders ofsyncytia; (FIG. 6C) the extent of pore formation determined byco-transfection of GFP with pcDNA3, p14V9T, or endV9T at a 1:4 ratio.Results are shown as means±standard error of values obtained from dotplots (solid bars) adjusted to subtract background pcDNA3 pore formationor Overton subtractions (empty bars) from three experiments done intriplicate;

(FIG. 7A) is a schematic representation of the p15 protein indicatingthe location and sequence of the HP, spanning residues 68-87, inclusive.“ecto” represents ectodomain, “TM” represents transmembrane domain,“endo” represents “endodomain”, “+++” represents polybasic region and“HP” represents hydrophobic patch (SEQ ID NO: 8); (FIG. 7B) is agraphical representation of QM5 cells transfected with p15 or the HPscan mutants and the average number of syncytial nuclei per field asdetermined from Giesma stained monolayers 8 hours post-transfection. Theresidues substituted by alanines in each construct are indicated.Results are expressed as means±standard error of an experiment donethree times in triplicate; (FIG. 7C) is a graphical representationshowing the extent of pore formation determined by co-transfection ofGFP with p15 or the alanine scan constructs at a 1:4 ratio. 10,000 gatedcells were counted and the percentage of GFP positive cells staining redis indicated. Results are shown as means±standard deviation of Overtonsubtractions obtained from two pooled experiments done in duplicate;

(FIG. 8A) is an immunoblot showing relative expression of p15 HP alaninescan mutants of lysates from transfected QM5 cells. Lane 1-p15, lane2-p15HP-LGL, lane 3-p15-LSY, lane 4-p15-GAG, lane 5-p15-VAS, lane6-p15-LPL, lane 7-p15-LNV and lane 8-p15-1A; (FIG. 8B) shows QM5 cellstransfected with C-terminally GFP-tagged p15G2A, p15-GAG or p15-LPL (notshown) or GFP as a soluble control, and chased to the cell surface witha 3 hour cycloheximide treatment (200 μg/mL), 20 hourspost-transfection. Arrows indicate regions of the plasma membrane inwhich protein has accumulated;

FIG. 9A and FIG. 9B are graphical representations showing (FIG. 9A) QM5cells transfected with p15, p15L80A, p15P81A or p15L82A. Results areexpressed as means±standard deviation of a representative experimentdone in triplicate; (FIG. 9B) Giesma stained cells transfected withp15P81A for 8 (a) or 24 (b) hours. Arrows indicate the borders ofsyncytia;

FIG. 10(A) is a schematic representation showing the linear amino acidsequences of synthetic peptides used to induce liposome-liposome fusion(SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10); (FIG. 10B) a graphicalrepresentation showing the ability of 50 μM of p15HP, p14myr+ or p14myr−synthetic peptides resuspended in DMSO, or the volume equivalent ofDMSO, to mediate lipid mixing of 100 μM of lipid (1:1:1,DOPC:DOPE:Chol); (FIG. 10C) is a graphical representation showing theability of 50, 25 or 10 μM of p15HPscr synthetic peptide resuspended inDMSO to induce lipid mixing; (FIG. 10D) is a graphical representationshowing the ability of 50, 25 or 10 μM of p15HPscr synthetic peptideresuspended in DMSO to induce lipid mixing;

(FIG. 11A) is a linear representation of the transmembrane domains ofthe p15 of the present invention and deletion constructs (p15Δ21,p15Δ21/22 and p15Δ40-43) or N-terminal substitution constructs (p15I21Aand p15I21/22A) (SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ IDNO:14, SEQ ID NO: 15 and SEQ ID NO:16). (FIG. 11B) represents QM5 cellstransfected with authentic p15 (a), p15Δ21 (b), p15Δ21/22 (c) orp15Δ40-43 (d) then methanol fixed and Giemsa-stained 10 hrpost-transfection to detect syncytia formation (indicated by arrows inpanel a). Scale bars=100 μm (FIG. 11C) represents QM5 cells transfectedwith p15I21A (a) or p15I21/22A (b) and immunostained 24 hpost-transfection using polyclonal anti-p15. Arrows indicateantigen-positive single cell foci;

(FIG. 12A) represents transfected QM5 cells lysates (L) werefractionated soluble (S) and membrane (M) fractions byultracentrifugation. The presence of p15, p15Δ21, p15Δ21/22 or p15Δ40-43in each fraction was detected by SDS-PAGE and immunoblotting usingpolyclonal anti-p15 C-terminal antiserum. (FIG. 12B) represents QM5cells were transfected with C-terminally GFP-tagged p15Δ21, p15Δ21/22 orp15Δ40-43 and chased to the cell surface with a 3 hr cycloheximidetreatment at 20 h post-transfection, then fixed with formaldehyde.Images shown represent two or three merged fluorescent Z sections (leftpanels) overlaid on the DIC image (right panels). Arrows indicate plasmamembrane-localized p15. Scale bars=20 μm;

(FIG. 13(A) is a linear representation of the TMDs of authentic p15 andthe β-branched substitution constructs (121T, IV21/22T, I21L andIV21/22L) (SEQ ID NO:11). (FIG. 13B) is a graphical representationshowing the average number of syncytial nuclei present in QM5 cellstransfected with p15, p15I21L or p15I21T at 6 to 16 h post-transfectionwas quantified by microscopic examination of five random fields ofGiemsa stained monolayers. The number of syncytial nuclei induced byauthentic p15 exceeded countable levels after 8 h post-transfection.Results are the mean±S.D. from a representative experiment done intriplicate. (FIG. 13C) shows lysates from QM5 cells transfected with p15or the indicated point mutant were analyzed by SDS-PAGE andimmunoblotting using polyclonal anti-p15 C26 term, or anti-actin as aloading control. (FIG. 13D) QM5 cells transfected with the indicatedβ-branched substitution constructs were assessed for cell-cell poreformation at 9 h post-transfection using a dual color FACS-basedfluorescent pore formation assay. Results are the mean±S.D. from arepresentative experiment done in triplicate indicating the percent ofdonor cells co-expressing EGFP and the indicated p15 construct thatacquired the small, aqueous calcein red fluorescent cytoplasmic markerfrom target cells;

(FIG. 14A) is a linear representation of the TMD of wild-type p15 andthe locations of alanine substitutions of the glycine and serineresidues in the indicated substitution constructs (SEQ ID NO:11). (FIG.14B) is a graphical representation showing the percent syncytiumformation relative to authentic p15 at 10 hr post-transfection wasdetermined by comparing the average number of syncytial nuclei per fieldin Giemsa-stained QM5 monolayers transfected with the indicated glycineand serine point substitutions. Results are the mean±S.D. from arepresentative experiment done in triplicate;

(FIG. 15A) represents a schematic of the pFastBec-p14-bombesinconstruction; (FIG. 15B) is an immunoblot showing expression of therecombinant virus in sf21 cells and verified protein production;

FIG. 16 represents a linear amino acid sequence and correspondingnucleic acid sequence of a recombinant polypeptide of the presentinvention (SEQ ID NO:17 and SEQ ID NO:18);

FIG. 17A and FIG. 17B show immunofluorescence images (FIG. 17A) ofHT1080 td tomato cells transfected with pFastBac plasmids containingempty vector, p14 or p14-bombesin, and quantitative analysis (FIG. 17B)of syncytia formation using flow cytometry;

FIG. 18 shows immunofluorescence images of syncytia formation in QM5quail muscle cells incubated with purified protein (p14 andp14-bombesin). Cells are stained with CellTracker Green and DAPI (blue);

FIG. 19 shows fluorescence imaging of PC-3 prostate cancer cellsincubated with liposomes containing FITC-dextran cargo. FITC-dextran(green), DAPI (blue) and actin cytoskeleton (yellow);

FIG. 20 is a graphical representation of the fluorescence intensity ofPC-3 prostate cancer cells exposed to p14-bombesin liposomes containingfluorescent dextran;

FIG. 21 represents siRNA electrophoresis for loading quantification;

FIG. 22A and FIG. 22B are immunofluorescence images (FIG. 22A) andgraphical representations (FIG. 22B) of Cy5-siRNA accumulation in HT1080cells 5 mins, 30 mins and 24 hours after exposure to Cy5-siRNAcontaining liposomes having fusogenic proteins embedded in theirmembranes compared to delivery by standard liposomes; and

FIG. 23A is a graphical representation and FIG. 23B is animmunofluorescence image of GFP expression in HT1080 cells followingdelivery of siRNA using liposomes having fusogenic proteins embedded intheir membrane compared to delivery by standard liposomes.

DESCRIPTION OF THE INVENTION

The following description is of a preferred embodiment by way of exampleonly and without limitation to the combination of features necessary forcarrying the invention into effect.

The recombinant polypeptide of the present invention comprises asequence corresponding to the ectodomain of p14 fusion-associated smalltransmembrane (FAST) protein, a transmembrane domain from a FAST proteinand the endodomain of p15 FAST protein (FIG. 1A). In the case of FIG.1A, the transmembrane domain comes from the p15 FAST protein. However,as described below, the transmembrane domain can come from any FASTprotein, such as p14, p10 or p15. Preferably, the transmembrane domaincomprises 23 amino acid residues, at least two hydrophobic, β-branchedresidues adjacent the ectodomain, three consecutive serine residuesimmediately adjacent the at least two hydrophobic, β-branched residues,and a glycine residue at positions 7 and 13 from the junction betweenthe ectodomain and the first hydrophobic, β-branched residue. Thispolypeptide has been shown to increase syncytia formation compared towild-type p14. This increase in syncytia formation also correlated withan increase in pore formation.

By “polypeptide” or “protein” is meant any chain of amino acids,regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation). Both terms are used interchangeablyin the present application.

In one aspect of the invention, proteins are provided which are encodedby the genome of Reoviridae, and whose amino acid sequence is free offusion peptide motifs.

The family Reoviridae includes the genus Orthoreovirus, which includesavian, mammalian and reptilian reoviruses, as well as the genusAquareovirus.

The ectodomain or extracellular region of the p14 FAST protein is shownin FIG. 1B. As discussed below with respect to the transmembrane domainsof the invention, the precise boundary of the p14 ectodomain isvariable. As such, the last two residues of the p14 ectodomain [WE]could therefore be part of the transmembrane domain. The polynucleotidesequence of FIG. 1E includes codons corresponding to the WE amino acids.The composition of the p14 ectodomain has been described elsewhere (seeCorcoran and Duncan, J Virol 78: 4342-4351, 2004; Corcoran et al., JBiol Chem 279:51386-51394, the contents of which are herein incorporatedby reference). The p14 ectodomain contains a myristoylation motif, whichis maintained in the recombinant polypeptide of the present invention.

In one embodiment, the myristoylation consensus sequence is: (initiatorMet removed) Gly1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-, where AA2, AA3, AA4,AA5, AA6 are small uncharged residues, AA3 and AA4 are preferablyneutral, where AA5 is preferably serine or threonine, where AA6 is notproline, and where AA7 and AA8 are preferably basic (Towler et al.,Annu. Rev. Biochem. 57:69-99 (1998); Resh, Biochim. Biophys. Acta.1451:1-16 (1999), the contents of which are herein incorporated). Inanother embodiment, the myristoylation consensus sequence is G S G P S NF V (SEQ ID NO:19).

It is contemplated that sequence variation can occur between afunctional p14 ectodomain and the wild-type sequence. For example,substitutions such as V9I, P 13A, G14A and E15A maintain fusion activity(Corcoran et al., J Biol Chem 279:51386-51394). As shown in FIG. 5A-FIG.5D and FIG. 6A-FIG. 6C, when amino acids 1-21 of the p14 ectodomain areoccupied by an anti-p 14ecto antibody, or when amino acid 9 of the p14ectodomain is changed from valine to threonine, the recombinantpolypeptide of the present invention is able to retain its ability toform syncytia. As such, variation in the wild-type p14 ectodomainsequence can occur while maintaining functionality of the recombinantpolypeptide described herein.

As would be understood by a person skilled in the art, percent identityis measured using sequence analysis software such as the SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705. Amino acid sequences are aligned to maximize identity. Gaps maybe artificially introduced into the sequence to attain proper alignment.Once the optimal alignment has been set up, the degree of identity isestablished by recording all of the positions in which the amino acidsof both sequences are identical, relative to the total number ofpositions.

The proteins of the invention are structurally defined in that they areeither encoded by Reoviridae are related to Reoviridae fusion proteinsmainly at the transmembrane domain. Preferably the transmembrane domaincomprises 23 amino acid residues, at least two hydrophobic, β-branchedresidues adjacent the ectodomain, three consecutive serine residuesimmediately adjacent the at least two hydrophobic, β-branched residues,and a glycine residue at positions 7 and 13 from the junction betweenthe ectodomain and the first hydrophobic, β-branched residue.

The at least two hydrophobic, β-branched residues can be eitherisoleucine and valine. Preferably, the N-terminal hydrophobic,β-branched residue is isoleucine followed by valine.

Preferably, the transmembrane domain is represented by the sequenceshown in FIG. 11A-FIG. 11C. However, other than the above-describedconsiderations, variability from the sequence shown in FIG. 11A iscontemplated.

The endodomain or cytoplasmic region of the p15 FAST protein is shown inFIG. 1C. The p15 endodomain comprises a hydrophobic patch, which istypically found on ectodomains, or extracellular regions, of many fusionproteins. Hydrophobicity values are determined according to methodsknown in the art. According to the present invention, the values arecalculated using the normalized hydrophobicity scale of Eisenberg, Ann.Rev. Biochem., 53:595-623, 1984.

The p15 endodomain hydrophobic patch is a short sequence of about 20residues containing an unusually high proportion of glycine and alanine.For example, 30% of the hydrophobic patch contains glycine or alanine.Within the hydrophobic patch, two glycine and one proline residues arerequired for syncytia and pore formation in the recombinant polypeptide.In addition to the hydrophobic patch, a polybasic region exists in astretch of 40 residues that includes the hydrophobic patch.

One aspect of the invention provides sequences that are identical orsubstantially identical to the sequences of FIG. 1A-FIG. 1F. By “aminoacid sequence substantially identical” is meant a sequence that is atleast 80%, preferably 90%, more preferably 95% identical to an aminoacid sequence of reference and that preferably differs from the sequenceof reference by a majority of conservative amino acid substitutions.

Conservative amino acid substitutions are substitutions among aminoacids of the same class. These classes include, for example, amino acidshaving uncharged polar side chains, such as asparagine, glutamine,serine, threonine, and tyrosine; amino acids having basic side chains,such as lysine, arginine, and histidine; amino acids having acidic sidechains, such as aspartic acid and glutamic acid; and amino acids havingnonpolar side chains, such as glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan and cysteine.

Proteins having a sequence homologous to the sequences of FIG. 1A-FIG.1F include naturally occurring allelic variants, as well as mutants orany other non-naturally occurring variants that retain the membranefusion properties of the polypeptides of the sequences of FIG. 1A-FIG.1F. As is known in the art, an allelic variant is an alternate form of apolypeptide that is characterized as having a substitution, deletion, oraddition of one or more amino acids that retain the biological functionof the polypeptide, i.e., the membrane-fusion activity.

Homologs and fragments thereof that do not occur naturally are designedusing known methods for identifying regions of the protein that arelikely to tolerate amino acid sequence changes and/or deletions. As anexample, homologous polypeptides from different species are compared;conserved sequences are identified. The more divergent sequences are themost likely to tolerate sequence changes. Percent identity andsimilarity among sequences may be analyzed using, as an example, theBLAST homology searching algorithm of Altschul et al., Nucleic AcidsRes. 25:3389-3402, 1997. Alternatively, a particular amino acid residueor sequence within the polypeptide can be mutated in vitro, then themutant polypeptides screened for their ability to promote membranefusion.

According to one aspect of the invention, isolated polynucleotides areprovided which encode the membrane fusion proteins of the invention. Inone embodiment, the polynucleotides are those shown in FIG. 1A-FIG. 1F.However, a person skilled in the art would readily understand andappreciate that redundancy in the genetic code allows for some variationin the actual polynucleotide sequences that encodes for the membranefusion proteins of the invention. For example, the standard genetic codeprovides that the codons: CCT, CCC, CCA and CCG encode for proline.

The term “isolated polynucleotide” is defined as a polynucleotideremoved from the environment in which it naturally occurs. For example,a naturally-occurring DNA molecule present in the genome of a livingvirus or as part of a gene bank is not isolated, but the same moleculeseparated from the remaining part of the viral genome, as a result of,e.g., a cloning event (amplification), is isolated. Typically, anisolated polynucleotide molecule is free from polynucleotide regions(e.g., coding regions) with which it is immediately contiguous at the 5′or 3′ end, in the naturally occurring genome. Such isolatedpolynucleotides may be part of a vector or a composition and still bedefined as isolated in that such a vector or composition is not part ofthe natural environment of such polynucleotide.

The polynucleotide of the invention is either RNA or DNA (cDNA, genomicDNA, or synthetic DNA), or modifications, variants, homologs orfragments thereof. The DNA is either double-stranded or single-stranded,and, if single-stranded, is either the coding strand or the non-coding(anti-sense) strand. Any one of the sequences that encode the proteinsof the invention as shown in FIG. 1A-FIG. 1F is (a) a coding sequence,(b) a ribonucleotide sequence derived from transcription of (a), or (c)a coding sequence which uses the redundancy or degeneracy of the geneticcode to encode the same polypeptides.

Homologous polynucleotide sequences are defined in a similar manner tohomologous amino acid sequences. Preferably, a homologous polynucleotidesequence is one that is at least 45%, more preferably 60%, and mostpreferably 85% identical to sequence encoding the proteins of theinvention, or to the coding sequences of the sequences shown in FIG.1D-FIG. 1F, or to the sequence encoding the proteins shown in FIG. 1C.

Polynucleotides encoding homologous polypeptides or allelic variants areretrieved by polymerase chain reaction (PCR) amplification of genomicviral polynucleotides extracted by conventional methods. This involvesthe use of synthetic oligonucleotide primers matching upstream anddownstream of the 5′ and 3′ends of the coding region.

Suitable primers are designed according to the nucleotide sequenceinformation provided in FIG. 1A-FIG. 1F. The procedure is as follows: aprimer is selected which consists of 10 to 40, preferably 15 to 25nucleotides. It is advantageous to select primers containing C and Gnucleotides in a proportion sufficient to ensure efficienthybridization; i.e., an amount of C and G nucleotides of at least 40%,preferably 50% of the total nucleotide content. A standard PCR reactioncontains typically 0.5 to 5 Units of Taq DNA polymerase per 100 gel, 20to 200 pM deoxynucleotide each, preferably at equivalent concentrations,0.5 to 2.5 mM magnesium over the total deoxynucleotide concentration,10⁵ to 10⁶ target molecules, and about 20 pmol of each primer. About 25to 50 PCR cycles are performed, with an annealing temperature 15° C. to5° C. below the true Tm of the primers. A more stringent annealingtemperature improves discrimination against incorrectly annealed primersand reduces incorporation of incorrect nucleotides at the 3′ end ofprimers. A denaturation temperature of 95° C. to 97° C. is typical,although higher temperatures may be appropriate for denaturation ofG+C-rich targets. The number of cycles performed depends on the startingconcentration of target molecules, though typically more than 40 cyclesis not recommended as non-specific background products tend toaccumulate.

An alternative method for retrieving polynucleotides encoding homologouspolypeptides or allelic variants is by hybridization screening of a DNAor RNA library. Hybridization procedures are well-known in the art andare described in Ausubel et al., (Current Protocols in MolecularBiology, John Wiley & Sons Inc., 1994), Silhavy et al., (Experimentswith Gene Fusions, Cold Spring Harbor Laboratory Press, 1984), and Daviset al., (A Manual for Genetic Engineering: Advanced Bacterial Genetics,Cold Spring Harbor Laboratory Press, 1980). Important parameters foroptimizing hybridization conditions are reflected in a formula used toobtain the critical melting temperature above which two complementaryDNA strands separate from each other (Casey and Davidson, Nucl. AcidsRes., 4:1539, 1977). For polynucleotides of about 600 nucleotides orlarger, this formula is as follows: T_(m)=81.5+0.41×(% G+C)+16.6 log(cation ion concentration)−0.63×(% formamide)−600/base number. Underappropriate stringency conditions, hybridization temperature (T_(h)) isapproximately 20 to 40° C., 20 to 25° C., or, preferably 30 to 40° C.below the calculated T_(m).

Those skilled in the art will understand that optimal temperature andsalt conditions can be readily determined.

Polynucleotide molecules according to the invention, including RNA, DNA,or modifications or combinations thereof, have various applications. ADNA molecule is used, for example, (i) in a process for producing theencoded polypeptide in a recombinant host system, (ii) as part of a genedelivery system, e.g., liposomes, which, upon delivery, becomesexpressed and promote membrane fusion, (iii) operably linked toregulatory elements as part of an expression cassette which, when turnedon, expresses the polynucleotide and promote membrane fusion, and, (iv)as a probe or primer.

Accordingly, one aspect of the invention encompasses (i) an expressioncassette containing a polynucleotide of the invention placed under thecontrol of the elements required for expression, in particular under thecontrol of an appropriate promoter; (ii) an expression vector containingan expression cassette of the invention; (iii) a procaryotic oreucaryotic cell transformed or transfected with an expression cassetteand/or vector of the invention, as well as (iv) a process for producinga polypeptide or polypeptide derivative encoded by a polynucleotide ofthe invention, which involves culturing a procaryotic or eucaryotic hostcell transformed or transfected with an expression cassette and/orvector of the invention, under conditions that allow expression of theDNA molecule of the invention without being toxic to the host cell and,recovering the encoded polypeptide or polypeptide derivative from thehost cell culture.

A recombinant expression system is selected from procaryotic andeucaryotic hosts. Since the proteins of the invention promote membranefusion, host cells ale selected which can be maintained and which canexpress the proteins within tolerable limits of toxicity. Eucaryotichosts include yeast cells (e.g., Saccharomyces cerevisiae or Pichiapastoris), plant cells, and cells which preferably have a cell wall sothat the integrity of the host cell is not affected by the fusionactivity. A preferred expression system is a procaryotic host such as E.coli. Bacterial and eucaryotic cells are available from a number ofdifferent sources including commercial sources to those skilled in theart, e.g., the American Type Culture Collection (ATCC; Manassas, Va.,USA). Commercial sources of cells used for recombinant proteinexpression also provide instructions for usage of the cells.

One skilled in the art would readily understand that not all vectors andexpression control sequences and hosts would be expected to expressequally well the polynucleotides of this invention. With the guidelinesdescribed below, however, a selection of vectors, expression controlsequences and hosts may be made without undue experimentation andwithout departing from the scope of this invention.

In selecting a vector, the host must be chosen such that it is notaffected by the fusion activity of the expressed membrane fusionprotein. In addition, a host must be chosen that is compatible with thevector which is to exist and possibly replicate in it. Considerationsare made with respect to the vector copy number, the ability to controlthe copy number, expression of other proteins such as antibioticresistance. In selecting an expression control sequence, a number ofvariables are considered. Among the important variables are the relativestrength of the sequence (e.g., the ability to drive expression undervarious conditions), the ability to control the sequence's function,compatibility between the polynucleotide to be expressed and the controlsequence (e.g., secondary structures are considered to avoid hairpinstructures which prevent efficient transcription). In selecting thehost, unicellular hosts are selected which are: compatible with theselected vector, tolerant of any possible toxic effects of the expressedproduct, able to express the product efficiently, able to express theproduct in the desired conformation, easily scaled up, and easy to usefor purifying the final product.

The choice of the expression cassette depends on the host systemselected as well as the features desired for the expressed polypeptide.Typically, an expression cassette includes a promoter that is functionalin the selected host system and can be constitutive or inducible; aribosome binding site; a start codon (ATG) if necessary; a regionencoding a signal peptide; a polynucleotide of the invention; a stopcodon; and optionally a 3′ terminal region (translation and/ortranscription terminator). The signal peptide encoding region isadjacent to the polynucleotide of the invention and placed in properreading frame. The signal peptide-encoding region is homologous orheterologous to the DNA molecule encoding the mature polypeptide and iscompatible with the secretion apparatus of the host used for expression.The open reading frame constituted by the DNA molecule of the invention,solely or together with the signal peptide, is placed under the controlof the promoter so that transcription and translation occur in the hostsystem.

Promoters and signal peptide encoding regions are widely known andavailable to those skilled in the art and include, for example, thepromoter of Salmonella typhimurium (and derivatives) that is inducibleby arabinose (promoter araB) and is functional in Gram-negative bacteriasuch as E. coli [as described in U.S. Pat. No. 5,028,530 and in Cagnonet al. (Protein Eng., 4(7):843, 1991)]; the promoter of the gene ofbacteriophage T7 encoding RNA polymerase, that is functional in a numberof E. coli strains expressing T7 polymerase (described in U.S. Pat. No.4,952,496); OspA lipidation signal peptide; and RlpB lipidation signalpeptide (Takase et al., J Bacterial., 169:5692, 1987).

Promoters contemplated for use herein include inducible (e.g., minimalCMV promoter, minimal TK promoter, modified MMLV LTR), constitutive(e.g., chicken alpha-actin promoter, MMLV LTR (non-modified), DHFR),and/or tissue specific promoters.

Inducible promoters contemplated for use in the practice of the presentinvention comprise transcription regulatory regions that functionmaximally to promote transcription of mRNA under inducing conditions.Examples of suitable inducible promoters include DNA sequencescorresponding to: the E. coli lac operator responsive to IPTG (seeNakamura et al., Cell, 18:1109-1117, 1979); the metallothionein promotermetal-regulatory-elements responsive to heavy-metal (e.g., zinc)induction (see Evans et al., U.S. Pat. No. 4,870,009), the phage T7lacpromoter responsive to IPTG (see Studier et al., Methods Enzymol.,185:60-89, 1990; and U.S. Pat. No. 4,952,496), the heat-shock promoter;the TK minimal promoter; the CMV minimal promoter; a synthetic promoter;and the like.

The expression cassette is typically part of an expression vector, whichis selected for its ability to replicate in the chosen expressionsystem. Expression vectors (e.g., plasmids or viral vectors) can bechosen, for example, from those described in Pouwels et al., (CloningVectors: A Laboratory Manual 1985, Supp. 1987). Suitable expressionvectors can be purchased from various commercial sources.

Methods for transforming/transfecting host cells with expression vectorsare well-known in the art and depend on the host system selected asdescribed in Ausubel et al., (Current Protocols in Molecular Biology,John Wiley & Sons Inc., 1994).

Upon expression, a recombinant polypeptide of the invention (or apolypeptide derivative) is produced and remains in the intracellularcompartment, is secreted/excreted in the extracellular medium or in theperiplasmic space or is embedded in the cellular membrane. Thepolypeptide is recovered in a substantially purified form from the cellextract or from the supernatant after centrifugation of the recombinantcell culture. Typically, the recombinant polypeptide is purified byantibody-based affinity purification or by other well-known methods thatcan be readily adapted by a person skilled in the art, such as fusion ofthe polynucleotide encoding the polypeptide or its derivative to a smallaffinity binding domain. Antibodies useful for purifying byimmunoaffinity the polypeptides of the invention are obtained throughmethods known in the art.

The recombinant polypeptide described above can also be linked to atargeting ligand. Alternatively, a fusion-associated small transmembrane(FAST) protein can be linked to a targeting ligand to facilitate bindingof the liposome containing the FAST protein/targeting ligand to aspecific cell type.

FAST proteins have been described elsewhere, see, for example, U.S. Pat.No. 7,851,595 to Duncan (the contents of which are specificallyincorporated herein in its entirety by express reference thereto).Generally, the FAST proteins are membrane fusion proteins whose aminoacid sequences are free of any fusion peptide motifs and which arerelated to Reoviridae in that they comprise an amino acid sequence whichhas at least 33% overall identity to the membrane fusion protein encodedby Reoviridae, and comprise a transmembrane domain whose amino acidsequence has at least 60% amino acid sequence identity to thetransmembrane domain of the membrane fusion protein encoded byReoviridae. In this case, fusion peptide motifs can be either be thetype I or type II motifs. The fusion peptide motif (type I) is an aminoacid sequence typically 17 to 28 residues long, with a hydrophobicity ofabout 0.6 to 0.7, and whose content of alanine plus glycine is about 29to 43%. A fusion peptide motif (type II) is 16 to 20 residues long, hasa hydrophobicity value about 0.3 to 0.4, has an alanine plus glycinecontent of about 29 to 43%; and contains a heptad repeat.

The FAST proteins are encoded by, or related to, the genome ofOrthoreovirus or Aquareovirus, or the genome of a reovirus whichnaturally infects a poikilothermic host. Furthermore, the proteins ofthe invention are membrane fusion proteins whose amino acid sequencesare free of any fusion motif, as defined above, and which are related toOrthoreovirus or Aquareovirus in that they comprise an amino acidsequence which has at least 33% overall identity to the membrane fusionprotein encoded by Orthoreovirus or Aquareovirus, and comprise atransmembrane domain whose amino acid sequence has at least 60% aminoacid sequence identity to the transmembrane domain of the membranefusion protein encoded by Orthoreovirus or Aquareovirus.

As mentioned above, the recombinant polypeptide comprises a targetingligand. For the purposes of the present invention, the targeting ligandrefers to a polypeptide that is capable of recognizing and binding acell surface molecule to facilitate the interaction between the cell andthe liposome. For example, cells expressing gastrin-releasing peptide(GRP) receptors can be recognized by the targeting ligand, bombesin[Anastasi, A et al., Experientia, 27:166 (1971)]. GRP receptors havebeen shown to be expressed on the cell surface membranes of prostatecancer cells, but absent from normal prostate cells. This uniqueexpression profile makes bombesin a preferred targeting ligand for thedelivery of a substance encapsulated in liposome, such as achemotherapeutic agent, to a cancerous prostate cell. Since normal,healthy prostate cells do not express GRP receptors, delivery of thechemotherapeutic agent to these cells will be limited.

Four GRP receptor sub-types have been identified to date: GRP-R, NMB-R,BRS-3 and BB4-R. A very potent universal pan-bombesin ligand with astructure of [D-Tyr6, β-Alai 1, Phe13, Nle14] bombesin-(6-14) has beenshown to bind to all four GRP receptor subtypes with high affinity[Reubi, J C et al., Clin. Cancer Res., 8:1139 (2002)].

A variety of targeted ligands have been described in the art. Theseinclude both peptides capable of being recognized by cell surfacereceptors, as well as antibodies that recognize and bind to cell surfacereceptors. Further examples of such targeting ligands include:protease-cleaved collagen IV (TLTYTWS); PC3 target (8-3) (DTDSHVNL); PC3target (9-8)(DVVVALSDD); Linear RGDx1 (GRGDSG); and Linear RGDx3(GRGDSGGRGDSGGRGDSG).

The use of liposomes having fusogenic proteins, as described herein,embedded in their membrane to deliver a payload, such as a bioactivecompound, have been shown to be more effective in delivering the payloadto a cell. For example, liposomes carrying an siRNA payload and havingfusogenic proteins as described herein embedded in their membrane havebeen shown to more effectively reduce the expression of the targetedgene in a recipient cell compared to liposomes having no such fusogenicproteins in their membrane.

Various liposome compositions are known in the art. For example, neutralor anionic liposomes, and cationic lipids are known to facilitatedelivery of substance(s) to a host cell. These compounds are readilyavailable to one skilled in the art; for example, see Liposomes: APractical Approach, RCP New Ed, IRL press (1990).

Anionic and neutral liposomes are well-known in the art (see, e.g.,Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for adetailed description of methods for making liposomes) and are useful fordelivering a large range of products, including polynucleotides, such assiRNA.

Cationic lipids are also known in the art and are commonly used for drugor gene delivery. Such lipids include Lipofectin™ also known as DOTMA(N-[1-(2,3-dioleyloxy) propyls-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives such as DC-Chol(3-beta-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP Pat. No.187,702, PCT Intl. Pat. Appl. Publ. No. WO 90/11092, U.S. Pat. No.5,283,185, PCT Intl. Pat. Appl. Publ. No. WO 91/15501, PCT Intl. Pat.Appl. Publ. No. WO 95/26356, and U.S. Pat. No. 5,527,928. Cationiclipids for delivery of polynucleotides are preferably used inassociation with a neutral lipid such as DOPE (dioleylphosphatidylethanolamine), as described in PCT Intl. Pat. Appl. Publ.No. WO 90/11092 as an example.

Formulations containing cationic liposomes may optionally contain othertransfection-facilitating compounds.

A number of them are described in PCT Intl. Pat. Appl. Publ. Nos. WO93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They includespermine derivatives useful for facilitating the transport of DNAthrough the nuclear membrane (see, for example, PCT Intl. Pat. Appl.Publ. No. WO 93/18759) and membrane-permeabilizing compounds such asGALA, Gramicidine S, and cationic bile salts (see, for example, PCTIntl. Pat. Appl. Publ: No. WO 93/19768).

Unilamellar liposomes of the present invention can be prepared frommultilamellar lipid vesicles. For example, a mixture ofDOPC:COPE:cholesterol:DC-cholesterol in a molar ratio of 60:30:4:6 canbe used to prepare a multilamellar lipid vesicle suspension. However,lipid vesicles containing PE-PEG2000 and/or DOTAP may be required incertain circumstances. The multilamellar lipid vesicles can be preparedby a number of different methods, which will be known to a personskilled in the art, such as the thin film method described by Fenske andCullis, 2005, Methods Enzymol., 391:7-40.

Unilamellar liposomes can be extruded from the multilamellar lipidvesicle suspension by manual extrusion through a polycarbonate membraneof defined pore size, using gas-tight, glass syringes, such as theLiposoFast-Basic (Avestin, Inc., Ottawa, ON, CANADA). In this procedure,the sample is passed through the membrane by pushing the sample back andforth between two syringes. In most cases, a 50-nm diameter liposomewill allow for a sufficient amount of siRNA to be packaged in theliposome to decrease expression of the target gene. In order to achievea specific diameter of liposome, the multilamellar lipid vesiclesuspension is passed through a filter having a specific diameter of poresize.

In one embodiment, the unilamellar liposomes are further processed togenerate a wrapped liposome or wrapsome. Yagi et al., in Cancer Res.2009, 69(16):6531-6538 describe a process for generating such wrapsomes.Briefly, a wrapsome core is generated using 100% DOTAP andreconstitution with a liposome buffer containing4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, sodium chloride andphosphate buffered saline. The multilamellar wrapsome core is thenfurther processed to isolate unilamellar wrapsomes with a diameterapproximately equal to the diameter of the unilamellar liposomesdescribed above. Prior to encapsulating the unilamellar liposomesdescribed above with the wrapsome, siRNA is mixed with the unilamellarliposome to create an siRNA containing liposome. The wrapsomes are thendissolved in ethanol to create an envelope solution. This solution isthen added to the siRNA containing liposome, in the presence of theliposome buffer described above, causing the envelope to wrap around thesiRNA containing liposome to create a liposome capable of carrying apayload, such as siRNA to a cell.

The fusogenic proteins of the present invention can be inserted into theliposomal membrane by the detergent depletion method derived from theconcepts outlined previously in such publications as Rigaud et al.,1995, Biochim. Biophys. Acta, 1231(3):223-46; Rigaud and Levy, 2003,Methods Enzylmol., 372:65-86; and Top et al., 2005, EMBO J.,24(17):2980-8, the contents of which are incorporated herein byreference. Briefly, the purified fusogenic protein is reconstituted intothe liposomes by mixing the detergent-suspended fusogenic protein withliposomes pre-saturated with detergent, followed by removal of thedetergent.

To assist in the process of inserting the fusogenic protein into theliposome membrane, n-octyl β-D-glucopyranoside (OG) is used in thedetergent. Based on the size and type of liposomes used, an optimalconcentration of OG should be determined by incrementally adding OG to asolution of liposomal lipids across a 0-2.0% final volume concentrationspectrum. Absorbance is detected at λ600 nm and a plot derived ofabsorbance versus OG %. The optimal concentration is just below thecritical OG concentration which causes dissolution of the liposomes.

The liposomes of the present invention having fusogenic proteinsembedded in their membranes are used to encapsulate small-interferingRNA or siRNA nucleic acids. siRNAs have been used to switch off genes inmammalian cells without initiating an acute phase response, i.e., a hostdefense mechanism that often results in cell death (Caplen et al., PNAS98(17):9742-7, 2001; Elbashir et al., Methods 26(2):199-213, 2002).There is increasing evidence of post-transcriptional gene silencing byRNA interference (RNAi) for inhibiting targeted expression in mammaliancells at the mRNA level, in human cells. There is additional evidence ofeffective methods for inhibiting the proliferation and migration oftumor cells in human patients, and for inhibiting metastatic cancerdevelopment [see, e.g., Caplen et al. (supra)].

An siRNA is a nucleic acid that forms a double stranded RNA and has theability to reduce or inhibit expression of a gene or target gene whenthe siRNA is delivered to or expressed in the same cell as the gene ortarget gene. siRNA is short double-stranded RNA formed by thecomplementary strands. Complementary portions of the siRNA thathybridize to form the double stranded molecule often have substantial orcomplete identity to the target molecule sequence. In one embodiment, ansiRNA is a nucleic acid that has substantial or complete identity to atarget gene and forms a double stranded siRNA.

When designing the siRNA molecules, the targeted region is selected froma given DNA sequence beginning 50 to 100 nucleotides downstream of thestart codon. See, e.g., Elbashir et al., (supra). Initially, 5′ or 3′UTRs and regions nearby the start codon were avoided assuming thatUTR-binding proteins and/or translation initiation complexes mayinterfere with binding of the siRNP or RISC endonuclease complex.Sometimes regions of the target 23 nucleotides in length conforming tothe sequence motif AA (N19)TT (N, an nucleotide), and regions withapproximately 30% to 70% G/C-content (often about 50% G/C-content) oftenare selected. If no suitable sequences are found, the search often isextended using the motif NA (N21). The sequence of the sense siRNAsometimes corresponds to (N19) TT or N21 (position 3 to 23 of the 23-ntmotif), respectively. In the latter case, the 3′ end of the sense siRNAoften is converted to TT. The rationale for this sequence conversion isto generate a symmetric duplex with respect to the sequence compositionof the sense and antisense 3′ overhangs. The antisense siRNA issynthesized as the complement to position 1 to 21 of the 23-nt motif.Because position 1 of the 23-nt motif is not recognizedsequence-specifically by the antisense siRNA, the 3′-most nucleotideresidue of the antisense siRNA can be chosen deliberately. However, thepenultimate nucleotide of the antisense siRNA (complementary to position2 of the 23-nt motif) often is complementary to the targeted sequence.For simplifying chemical synthesis, TT often is utilized. siRNAscorresponding to the target motif NAR (N17)YNN, where R is purine (A,G)and Y is pyrimidine (C,U), often are selected. Respective 21 nucleotidesense and antisense siRNAs often begin with a purine nucleotide and canalso be expressed from pol III expression vectors without a change intargeting site. Expression of RNAs from pol III promoters can be moreefficient when the first transcribed nucleotide is a purine.

The sequence of the siRNA can correspond to the full-length target gene,or a subsequence thereof. Often, the siRNA is about 15 to about 50nucleotides in length (e.g., each complementary sequence of the doublestranded siRNA is 15 to 50 nucleotides in length, and the doublestranded siRNA is about 15 to 50 base pairs in length, sometimes about20 to 30 nucleotides in length or about 20 to 25 nucleotides in length,e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength. The siRNA sometimes is about 21 nucleotides in length. Methodsof using siRNA are known in the art, and specific siRNA molecules may bepurchased from a number of companies including Dharmacon Research, Inc.

siRNA nucleic acids can be altered to form modified nucleic acidmolecules. The nucleic acids can be altered at base moieties, sugarmoieties or phosphate backbone moieties to improve stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of nucleic acid molecules can be modifiedto generate peptide nucleic acids (see Hyrup et al., Bioorg. MedicinalChem., 4(1): 5-23 (1996)).

One aspect of the invention provides methods of using the proteins,polynucleotides and compositions of the invention. Accordingly, methodsare provided to promote membrane fusion which comprise contacting themembranes to be fused with an effective amount of the above-describedproteins.

Membranes contemplated for fusion in accordance with the presentinvention include cell membranes, liposome membranes, proteoliposomemembranes, and the like.

In accordance with a still further embodiment of the present invention,there are provided methods for the production of liposome-liposomefusions or liposome-cell fusions, said methods comprising contactinglipids suitable for the formation of liposomes and a suitable cell inthe presence of one or more proteins as described herein.

In accordance with yet another embodiment of the present invention,there are provided improved methods for the intracellular delivery ofbioactive compounds employing liposomes, the improvement comprisingincorporating into said liposomes one or more proteins as describedherein.

The ability to promote efficient membrane fusion has broad applicabilityin clinical, industrial, and basic research situations. The recombinantproteins of the invention could be used as alternatives tochemically-induced membrane fusion to promote cell-cell fusion, forexample, during the production of hybridoma cells for monoclonalantibody production. In this instance, the reovirus fusion proteins andthe recombinant polypeptide thereof would be inducibly expressed frominside a transiently or permanently transfected cell population totrigger fusion of these cells with a target cell population.

The recombinant proteins also have application in enhancingliposome-cell fusion. Liposomes have been developed as a means tointroduce nucleic acids, proteins, and metabolic regulators into cells.Although liposome-cell fusion has been amply demonstrated, theunfavourable thermodynamics of membrane fusion contribute to variableefficiencies of fusion and cytotoxicity which lead to the development ofproteoliposomes-liposomes containing specific proteins to promote cellbinding and fusion.

Most of the proteoliposome studies reported in the art relate to the useof various enveloped virus fusion proteins. In accordance with thepresent invention, it is possible to take advantage of the novelstructural features associated with the recombinant proteins for use inproteoliposomes to enhance the intracellular delivery of bioactivecompounds (e.g., nucleic acids, proteins or peptides, pharmacologicalagents, and the like), both in cell culture and in vivo.

The recombinant proteins described herein have the ability to promotemembrane fusion in a diversity of cell types (e.g., fibroblasts andmacrophages) from different species (e.g., avian and mammalian,including human) suggesting limited cell receptor-specificity as well asthe general applicability of these proteins. It may also be possible totarget recombinant protein-containing proteoliposomes to specific celltypes by including specific receptor-binding proteins in the liposomemembrane. In this instance, the receptor-binding protein would confertargeted cell attachment of the liposome followed by subsequent enhancedliposome-cell fusion mediated by the recombinant protein.

The demonstrated ability of the p14 ectodomain/p15 endodomainrecombinant protein to induce cell-cell fusion indicates their potentialuse in the production of heterokaryons, for example, the generation ofhybridomas for monoclonal antibody production. The induction ofcell-cell fusion is usually triggered using the chemical fusogenpolyethylene glycol (PEG). Although this procedure does triggercell-cell fusion, toxic effects on cells hamper the efficiency ofheterokaryon isolation. It is generally believed that “natural” membranefusion is mediated by protein-lipid interactions, therefore,protein-mediated membrane fusion is likely to be much less cytotoxicthan chemically-induced cell fusion.

The demonstrated ability of the recombinant proteins to promoteefficient cell-cell fusion indicates their potential use as alternativesto chemical-induced cell fusion.

Expression of recombinant proteins in one population of cells, under thecontrol of a strong inducible promoter, could trigger fusion with asecond cell population, resulting in decreased cytotoxicity and moreefficient heterokaryon isolation. It is contemplated that therecombinant proteins of the invention could be added to cells in thepresence of a lipid carrier, such as Lipofectamine™, and inducecell-cell fusion and heterokaryon formation.

The recombinant proteins described herein represent alternatives to theuse of enveloped virus fusion proteins in the protein-mediatedenhancement of liposome-cell fusion for the intracellular delivery ofbioactive molecules. The potential advantages of the p14 ectodomain/p15endodomain recombinant proteins relate to their unique ability to morestrongly fuse to other cells compared to native reovirus proteins. Thelarge size, post-translational glycosylation, and complex tertiarystructure of the enveloped virus fusion proteins makes synthesis andpurification of the functional protein using recombinant DNA approachesand prokaryotic or eukaryotic expression systems problematic.

The majority of studies relating to the use of enveloped virus fusionproteins in proteoliposomes involve the production of virus particleswhich are subsequently purified, solubilized with detergent, and theviral envelopes containing the fusion protein are reconstituted into“virosomes” by removal of the detergent [see Grimaldi in Res. Virol.,146:289-293 (1995) and Ramani et al., FEBS Lett., 404:164-168 (1997)].

Unlike most of the enveloped virus fusion proteins, the reovirus fusionproteins, from which the recombinant proteins are derived, are smallmembrane proteins. Their small size and simple domain organizationsuggests that these proteins will be easier and more economical toproduce in a functional form using a diversity of expression andpurification protocols. It is also likely that the small size of therecombinant proteins contributes to less complex protein foldingpathways and tertiary structure required for correct proteinconformation. As a result, an increased diversity of extraction andsolubilization procedures (e.g., choice of detergents and denaturants)should be available to facilitate purification of the functional fusionprotein and incorporation into liposomes.

The attractive biological properties of the recombinant proteins relateto their pH-independent fusion mechanism with numerous cell types. Therecombinant proteins function at neutral pH, unlike the influenza virusHA protein, simplifying their use in cell culture and in vivo underphysiological conditions. Furthermore, the recombinant proteins fusenumerous types of cells suggesting their broad applicability asfusogens. This could include such primary cell types as dendritic cells,neurons, and stem cells which are difficult to transfect using standardtransfection reagents.

Accordingly, the p14 ectodomain/p15 endodomain recombinant proteinscould be used to promote liposome-cell fusion and the efficientintracellular delivery of DNA or other bioactive compounds into adiversity of cultured cell types, primary cell cultures, tissueexplants, or in vivo.

In order to use recombinant proteins for heterokaryon production, theproteins will need to be expressed in a controlled, inducible mannerfrom within cells using standard recombinant DNA approaches. The utilityof this approach has already been demonstrated in homologous cell-cellfusion in a non-inducible manner. In a similar fashion, these proteinscan promote cell-cell fusion between heterologous cell types in aninducible manner.

The development of recombinant proteins for enhanced liposome-cellfusion requires the expression and purification of the functional fusionproteins and their incorporation into liposome membranes to produceproteoliposomes. The recombinant proteins can be expressed and purifiedusing standard procedures. Expression can be accomplished employing avariety of expression systems, e.g., baculovirus or yeast eukaryoticexpression vectors or from prokaryotic expression vectors, depending onexpression levels and functional activity of the protein. Variousdetergent extraction procedures can be used to solubilize the proteins,which can then be purified as detergent-protein complexes using standardprotein purification protocols. The proteins are readily soluble invarious detergents (e.g., 0.8% Triton™ X100, 0.8% NP40, 0.8%octylglucoside) increasing the diversity of approaches available forfunctional protein purification. The small size of the recombinantproteins suggests that protein solubilization and purification should beconsiderably more simple than similar approaches to purify larger, morecomplex membrane proteins.

The detergent-protein complexes can be mixed with lipids and thedetergent removed by dialysis, chromatography, or extraction accordingto standard published procedures, similar to methods used to generateinfluenza HA or Sendai virus F protein-containing virosomes (seeGrimaldi, Res Virol, 146: 289-293, 1995 and Ramani et al., FEBS Lett,404: 164-168, 1997). These procedures will result in the production ofproteoliposomes, lipid vesicles containing the ARV, NBV, or BRV fusionproteins embedded in the vesicle membrane. Once again, optimalconditions for proteoliposome production can be empirically determinedas can the lipid composition and size of the proteoliposomes, which canaffect the efficiency of liposome-cell fusion. Bioactive molecules ofinterest (e.g., nucleic acids, proteins or peptides, pharmacologicalcompounds, and the like) can be included during the formation of theproteoliposmes to facilitate packaging of the molecule within theliposomes. The proteoliposomes can be purified by centrifugation andused to deliver bioactive molecules intracellularly, either in cellculture or in vivo, by protein-enhanced fusion of the proteoliposomeswith cell membranes.

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined by the claims.

Example 1: The p15 Endodomain Functions Modularly to Enhance MembraneFusion

Construct c78 is a truncation mutant in which 47 residues were deletedfrom the p14 C-terminus (Corcoran and Duncan, J. Virol., 78(8):4342-51,2004). As shown in FIG. 2, the c78p15 construct is a re-extension ofc78, in which the length of the cytoplasmic tail has been restored to125 residues using heterologous sequence corresponding to the last 47residues (93 to 140) of the p15 endodomain. These 47 C-terminal residuesare downstream of the p15 HP and thus the p15 HP is not included in thec78p15 chimeric construct. In agreement with earlier findings (Corcoranand Duncan, J. Virol., 78(8):4342-51, 2004), when a QM5 monolayer wastransfected with the p14 truncation mutant (c78), a loss of fusionactivity was observed by syncytial indexing (FIG. 3). When cells weretransfected with the re-extension construct c78p15 however, syncytiaformation was restored to authentic p14 levels (FIG. 3). In addition tothese truncation constructs, cells were also transfected with a chimericconstruct, p14end15, in which the entire endodomain of p14 was replacedby that of p15 (FIG. 3). In contrast to c78p15, the p14end15 constructincluded the p15 HP resulting in a chimera with two HPs; the exoplasmicHP of p14 and the cytoplasmic HP of p15.

When used to transfect a QM5 monolayer, the p14end15 construct inducedsyncytia formation to an extent that was greater than that of wild typep14 (FIG. 3).

To determine if syncytia formation correlated with pore formation,plasmids encoding authentic p14, c78, c78p15, p14end15 or the emptypcDNA3 vector were cotransfected into QM5 cells with the eGFP expressionplasmid and incubated to allow initial expression. At 3 hourspost-transfection, the transfected donor cells were overlaid with targetVero cells pre-labelled with aqueous calcein red dye. Cells wereco-incubated to allow for fusion then analyzed by flow cytometry for thetransfer of calcein dye to GFP-expressing cells. Uptake of calcein dyewas clearly detected in all transfected wells with the exception of thepcDNA3- and c78-transfected wells. The c78p15 construct, which inducedsyncytia formation to the same extent as wild type p14, also inducedpore formation to just slightly less that of p14 when analyzed by dotplot (FIG. 4A and FIG. 4B). When the same samples were analyzed byOverton subtractions however, c78p15 was found to induce pore formationto approximately the same extent as authentic p14 (FIG. 4B). Sincep14end15-transfected cells fuse so extensively, the lower levels of poreformation detected relative to the syncytial index may be due to amasking effect resulting from saturation of the assay detection limits.To test this hypothesis, the amount of p14end15 or p14 expressionplasmids co-transfected into donor cells was titrated in two-foldincrements, down to one eighth the amount transfected in panel B (FIG.4C). Plasmid titration corresponded to a decrease in fusion and unmaskedthe p14end15 fusion enhancement, revealing a clear increase in fusioninduced by p14end15 relative to p14 under less fusogenic conditions(FIG. 4C).

Example 2: The p15 Endodomain does not Alter Protein Expression Levelsat the Cell Surface

The FAST protein endodomains have recently been implicated a having arole in trafficking to the plasma membrane. Reduction in the levels ofprotein expressed on the cell surface are known to correlate to lowersyncytial indexes. Based upon these observations, experiments wereconducted to determine if the increased fusogenicity of p14end15 was dueto enhanced trafficking to the cell surface.

When comparing the surface levels of proteins in cells that have fusedto varying extents, syncytia formation may theoretically result in anincreased level of surface expression compared to non-fused cells due toan increase in the amount of translation machinery. Therefore, todirectly compare surface expression levels between constructs, theirfusion activity must be abolished to relieve the bias from syncytiaformation. In the case of constructs containing the ectodomain of p14,fusion can be inhibited using low dilutions of the anti-p14ecto antibody(FIG. 5A). Live monolayers are then stained with afluorescence-conjugated secondary antibody and surface expression can bequantified by flow cytometry. This approach was used in an attempt tocompare the levels of p14, c78, c78p15 and p14end15 present in theplasma membrane. While addition of the anti-p14ecto antibody inhibitedfusion mediated by p14 and c78p15 (data not shown), it was insufficientto completely inhibit fusion mediated by p14end15 (FIG. 5B). Although,antibody inhibition and subsequent surface staining did reveal thatc78p15 was expressed on the cell surface to a greater extent thanauthentic p14 (FIG. 5C). As such, increased surface expression levelsmay not always result in a corresponding increase in fusion.

To determine if p14end15 was present on the cell surface to the sameextent as wild type p14, a G2A substitution was made in the ectodomainwhich prevents N-terminal myristoylation. This mutation has beenpreviously shown to eliminate p14-mediated fusion activity withoutaffecting surface levels (Corcoran and Duncan, J. Virol., 78:4342-4351,2004). To measure surface levels of p14end15, QM5, monolayers wereco-transfected with empty pcDNA3 vector and the non-fusogenic p14G2A orp14end15G2A constructs (FIG. 5D). The amount of DNA used fortransfection was titrated to correspond to the amounts used in thedetermination of p14end15-induced pore formation (FIG. 4C). Whilep14end15 appeared to be present on the cell surface at slightly higherlevels than authentic p14 at the highest titration dose, surface levelsequalized with decreasing DNA dose (FIG. 5D).

At these lower transfection volumes, the greatest difference in fusionactivity between p14 and p14end15 could be observed. Therefore, thefusion potentiating activity of the p15 endodomain in the p14end15construct is likely due to a true enhancement of fusion enhancement, notan increase in cell surface protein levels.

Example 3: The p15 Endodomain can Rescue Fusion Defects in the p14Ectodomain

The anti-p14ecto antibody was raised against residues 1 to 36 of theauthentic p14 ectodomain and the p14 HP occupies residues 1-21 of theectodomain (FIG. 5A). As shown in FIG. 5B, incubation with anti-p14ectoantibody prevents syncytia formation in QM5 cells transfected with p14.Conversely, when QM5 cells transfected with the p14end15 construct wereincubated with anti-p14ecto antibody, syncytia formation still occurred.Levels of fusion are less than those induced by p14end15 in the absenceof the antibody.

The ability of p14end15 to overcome antibody inhibition also suggeststhat the motifs present in the p15 endodomain may be able to rescuefusion defects in the p14 ectodomain. Accordingly, the endV9T constructwas created in which a V9T substitution was made in the ectodomain HP ofp14end15 (FIG. 6A). The V9T substitution was previously found tocompletely abrogate the ability of authentic p14 to induce syncytiaformation (Corcoran et al., J Biol. Chem., 279(49):51386-94, 2004). Aswas the case with antibody inhibition, the V9T substitution did notresult in a loss of syncytia formation in a p14end15 backbone but dideliminate syncytiogenesis in a p14 backbone (FIG. 6B).

While p14V9T is non-fusogenic by syncytial indexing, when QM5 monolayerswere co-transfected with GFP and p14V9T or endV9T then co-incubated withcalcein-labelled target Vero cells to measure the extent of poreformation induced by these constructs, low levels of pore formationmediated by p14V9T were detectable by Overton subtraction, and to alesser extent by dot plot analysis (FIG. 6C). The endV9T constructmediated pore formation to a greater extent than p14V9T when analyzed byeither dot plot or histogram subtraction, supporting results obtained byGiemsa staining of transfected monolayers and suggesting that the p15endodomain may either enhance pore formation or stabilize the formationof fusion pores mediated by p14V9T.

A G2A substitution was generated in the p14end15 ectodomain, whichprevents myristoylation. This substitution does inhibitp14end15-mediated fusion (data not shown). This is in agreement with theobservation that a G2A substitution in the ectodomains of both p14 andp15 abrogates their fusion activity. Therefore, while the p15 endodomainis able to overcome defects in the HP of the p14 ectodomain, N-terminalmyristoylation still appears to be a requirement for p14end15-mediatedfusion.

Example 4: Glycine and Proline Residues in the p15 HP are Involved inFusion Activity

To determine if the p15 HP plays an enhancing role in thep14end15-mediated fusion reaction, an alanine scan of the HP was firstcarried out in the context of a wild type p15 backbone to identifyresidues involved in p15-mediated fusion (FIG. 7A). The residues of thep15 HP were substituted, three at a time, with alanine residues. Wherealanine was the naturally encoded residue (as is the case with thep15-GAG construct, for example), the alanine residue was maintained andnot replaced with a heterologous amino acid. By syncytial indexing oftransfected QM5 monolayers, two regions of the HP were identified to beinvolved in p15-mediated fusion (FIG. 7B). The p15 HP alanine scanmutants p15-GAG and p15-LPL were both found to be non-fusogenic bysyncytial indexing. This phenotype was next confirmed using thefluorescence-based pore formation assay described above and both mutantswere also found to be deficient in their ability to promote theformation of fusion pores (FIG. 7C).

When the presence of each mutant was tested in the lysates oftransfected QM5 cells by SDS-PAGE analysis and Western blotting, allseven alanine scan mutants could be detected. Equal amounts of lysates,based on total protein concentration, were loaded (FIG. 8A). Withapproximately equal amounts of actin, present in each sample as aloading control, both authentic p15 and p15-LGL (lanes 1 and 2,respectively) may have slightly elevated overall steady-state proteinlevels relative to the other alanine scan mutants. However, bothnon-fusogenic mutants, p15-GAG and p15-LPL, were clearly expressed intransfected lysates to approximately the same extent as the otheralanine scan mutants, which showed no fusion defects. In some instances,mutations of glycine and proline residues in fusion peptides can causetrafficking defects (Levy-Mintz and Kielian, J. Virol., 65(8):4292-300,1991; Shome and Kielian, Virology, 279(1):146-60, 2001), though thesedomains are found in the ectodomains of enveloped viral fusion proteins.

In addition, regions in the cytoplasmic domains of the FAST proteinshave been identified as being important for trafficking to the plasmamembrane. To determine if the GAG or LPL alanine substitutions resultedin a trafficking defect, p15G2A, p15-GAG and p15-LPL were C-terminallytagged with GFP and their presence on the surface of transfected cellswas detected. Cycloheximide was used to stop translation and deplete theintracellular pool of p15, chasing already translated proteins to thecell surface (FIG. 8B). While p15 and the p15 alanine scan constructswere both chased to the surface (p15 and p15-GAG shown), cycloheximidetreatment had no effect on cells expressing GFP (FIG. 8B). Takentogether, these observations indicate that the loss of fusion activityobserved in cells transfected with p15-GAG and p15-LPL is not due to atrafficking defect.

Since the alanine scan was conducted by substituting the HP residues inthe authentic p15 sequence in groups of three, experiments wereconducted to determine which of the three substituted residues ofp15-LPL were required for fusion activity. Single alanine substitutionswere made of the leucine and proline residues at positions 80-82 in thewild type p15 sequence to create p15L80A, p15P81A and p15L82A. QM5 cellswere transfected with each point substitution construct and a syncytialindex was taken at 8 hours post-transfection (FIG. 9A). While the L80Apoint substitution resulted in a limited loss of fusion activityrelative to wild type p15, p15L82A retained full fusion activity. Incontrast to the leucine point substitutions, no syncytia were formed inp15P81A-transfected monolayers at 8 hours post-transfection (FIG. 9A).However, if transfected cells are incubated under growth conditions for24 hours, limited p15P81A-induced syncyctia formation could be observed(FIG. 9B).

Example 5: A Peptide Corresponding to the p15 HP InducesLiposome-Liposome Lipid Mixing

To determine if the p15 HP has inherent membrane destabilizingproperties, the ability of a peptide corresponding to this region toinduce lipid mixing was investigated using a fluorescence resonanceenergy transfer (FRET) liposome-liposome lipid mixing assay.

Non-fluorescent liposomes and fluorescent liposomes composed ofDOPC:DOPE:Chol at a 1:1:1 molar lipid ratio were mixed with peptides andfusion was monitored as a loss of FRET and corresponding increase influorescence measured by fluorimetry. In the fluorescent liposomecomposition, 4% DOPE was replaced with 2% Rhodamine(Rho)DOPE and 2%NBD-DOPE. In addition to the sequence corresponding to the authentic p15HP sequence (p15HP), the previously described p14myr+ and p14myr− wereused as positive and negative controls for lipid mixing, respectively(FIG. 10A). When added to the liposome mixture, 50 μM p15HP inducedextensive lipid mixing greater than that induced by equal concentrationsof p14myr+. As expected, the p14myr− peptide did not induce lipid mixingabove background DMSO alone levels (FIG. 10B). When decreasing amountsof p15HP peptide were added to liposomes a dose-response in lipid mixingwas observed (FIG. 10C). Interestingly, when a scrambled version of thep15HP peptide, p15HPscr (FIG. 10A) was added to liposomes, this peptidealso induced a substantial loss of FRET (FIG. 10D). However, theincrease of fluorescence was not stable and the ability of the peptideto induce lipid mixing was not dose-dependent (FIG. 10D). Visually,addition of p15HPscr to liposomes resulted in increased turbidity of thesample which may represent the formation of liposome or peptideaggregates (data not shown). Since p15HPscr-induced loss of FRET did notappear to be dose-dependent it is possible that it resulted from anon-specific interaction of p15HPscr with liposomes. It is evidenthowever, that the sequence of the p15 HP has membrane interaction anddestabilization potential above and beyond that of the p14 ectodomain,which contains a fusion peptide.

Example 6: Truncation of the p15 TMD Eliminates Membrane Fusion Activity

The p14 and p10 TMDs are both predicted to be 19 residues in lengthwhile the p15 TMD is predicted to be 23 residues. To determine whetherthe terminal residues of the extended p15 TMD influence p15-inducedmembrane fusion, a series of deletions were created in which the p15 TMDwas truncated by either one or two residues from the N-terminal end(p15Δ21 and p15Δ21/22, respectively) or four residues from theC-terminal end (p15Δ40-43) (FIG. 11A). Giemsa stains of transfected QM5monolayers clearly revealed that each deletion of the authentic p15 TMDresulted in the complete loss of syncytiogenesis (FIG. 11B). Generally,when membrane fusion is lost due to insufficient transmembrane length,fusion of these mutants can be recovered by restoring the length of theTMD. The N-terminal isoleucine or isoleucine and valine residues thatwere deleted from the p15Δ21 and p15Δ21/22 constructs, were thereforereplaced with alanine residues to create p15I21A and p15IV21/22A,respectively (FIG. 11A). Replacement of the N terminal residues of thep15 TMD with alanine did not restore membrane fusion activity, withtransfected monolayers containing only individual antigen positive cells(FIG. 11C). The cell-cell fusion activity of the p15 FAST protein istherefore sensitive to either truncation or replacement of the terminalresidues of the TMD.

The FAST protein TMDs function as reverse signal-anchors to directco-translational membrane insertion. To determine whether the truncatedp15 TMDs could still function as reverse signal-anchors, transfectedcell lysates were separated into the cytoplasmic and membrane fractionsby high-speed ultracentrifugation. SDS-PAGE analysis and Westernblotting using anti-p15 C-terminal antiserum indicated each deletionconstruct was found exclusively in the pelleted membrane fraction (FIG.12A), confirming that the truncated TMDs were still capable of directingmembrane insertion of p15 as an integral membrane protein. Some viralfusion protein TMD truncation constructs also exhibit traffickingdefects. To assess whether truncation of the p15 TMD disruptedtrafficking to the plasma membrane, the p15Δ21, p15Δ21/22 and p15Δ40-43constructs were visualized by confocal immunofluorescent microscopy(FIG. 12B). In the absence of an antiserum that specifically recognizesthe p15 ectodomain, the p15 constructs were C-terminally tagged withGFP. To partially deplete intracellular pools of p15 (whose fluorescenceintensity masks the fluorescence of plasma membrane localized p15),cells were treated for 3 hrs with cycloheximide to stop translation andallow trafficking of the already translated p15 proteins to the cellsurface. A fusion-dead p15G2A myristoylation-minus construct was alsoemployed to allow extended incubations in the absence of syncytiumformation to improve detection of plasma-membrane localized p15. Thep15G2A mutant traffics to the cell surface to the same extent as wildtype p15, and this same procedure was previously used to demonstrate p15plasma membrane localization. As shown by confocal fluorescencemicroscopy, a clearly detected ring of fluorescence surroundingtransfected cells was evident in cells expressing authentic p15 and allof the truncated p15 TMD constructs (FIG. 12B), indicating truncation ofthe p15 TMD did not qualitatively interfere with p15 trafficking to theplasma membrane. The p15 TMD therefore specifically influences membranefusion activity, independent of the role of the TMD as a reversesignal-anchor or any role of the TMD in protein trafficking.

Example 7: The p15 TMD Requires Hydrophobic and D-Branched Residues atthe N-Terminus for Fusion

The truncation and alanine replacement results indicated the importanceof the N-terminal residues of the p15 TMD in membrane fusion activity.In addition to being hydrophobic, the N-terminal residues of the p15 TMDare also β-branched amino acids. β-branched residues have beenimplicated in the role of TMDs in membrane fusion, which relates totheir helix destabilizing activity and appears to be particularlyimportant when these residues are found at terminal positions in a TMD.To determine whether hydrophobicity and/or β-branched residues arerequired at the N-terminus of the p15 TMD, the terminal residues weresubstituted with either threonine, a β-branched and hydrophilic residue,or with leucine, a hydrophobic and non-β-branched residue (FIG. 13A). Toexamine the effect of these substitutions on membrane fusion, thekinetics of syncytium formation were obtained from transfected QM5monolayers that were fixed and Giemsa-stained from 6-16 hrspost-transfection (FIG. 13B). Authentic p15 rapidly induced cell-cellfusion with the onset of syncytia formation occurring within 4-6 hrspost-transfection. Robust fusion leading to apoptosis and disruption oftransfected monolayers induced by the authentic p15 protein precludedthe determination of a syncytial index past 8 h post-transfection.Substitutions of the N-terminal isoleucine residue with either threonineor leucine led to dramatic decreases in both the onset and extent ofsyncytiogenesis (FIG. 13B). At 8 hrs post-transfection, whenp15-transfected monolayers were approaching the maximal extent ofsyncytiogenesis, there was little if any evidence of syncytia in cellstransfected with p15I21T or p15I21L. However, syncytia became evidentshortly thereafter and continued to develop to significant levels by 16hrs post-transfection (FIG. 13B). Substitution of both the N-terminalisoleucine and valine residues resulted in a complete loss of syncytiumformation (data not shown). The decrease or loss of fusion activity withall of these constructs was not due to decreased expression of thesubstitution mutants, as determined by SDS-PAGE and Western blottinganalysis (FIG. 13C).

Syncytial indexing does not permit the detection of mutants for whichfusion is initiated but arrested prematurely at an earlier step, such ashemifusion or pore formation. Numerous studies have implicated the TMDsof enveloped viral proteins in the formation or expansion of stablefusion pores. To determine if the p15 TMD chimeras were capable ofinducing the formation of stable fusion pores, a previously describedFACS-based pore formation assay was used. QM5 cells were co-transfectedwith the pEGFP expression plasmid and a plasmid encoding eitherauthentic p15 or a p15 TMD substitution construct, and briefly incubatedto allow initial transgene expression. The transfected donor cells werethen overlaid with target Vero cells labeled with the aqueous fluorcalcein red. Donor and target cells were co-cultured to allow fusionevents to occur, then trypsin-treated to generate single cellsuspensions that were analyzed by flow cytometry. Quantifying thepercentage of GFP27 expressing donor cells that acquired the small 800Da calcein red fluor from target cells is indicative of stable poreformation. As with syncytium formation, by 9 hrs post-transfectionp15-transfected cells had induced extensive pore formation while the p15constructs containing N-terminal TMD substitutions induced no detectiblepore formation above background levels (data not shown). However,extended incubation of the constructs containing single N-terminal TMDsubstitutions eventually induced pore formation, the extent of whichgenerally paralleled their relative syncytiogenic capabilities, withp15I21T inducing more pore formation than p15I21L (FIG. 13D). Theconstructs containing double N-terminal substitutions that failed toinduce syncytium formation also failed to induce pore formation thatexceeded background levels (FIG. 13D). Therefore, replacement of theN-terminal, hydrophobic, β-branched isoleucine and valine residues witheither a polar β-branched residue or a hydrophobic non-β-branchedresidue dramatically impairs or eliminates p15 membrane fusion activityat, or prior to, the pore formation stage of the fusion reaction. Sincean alanine substitution of the same residue eliminated p15 fusionactivity (FIG. 11C), it is inferred that hydrophobic, β-branchedresidues at the N-terminus of the p15 TMD contribute to an active rolefor the TMD in membrane merger.

Example 8: Transmembrane Glycine and Serine Residues are Involved inp15-Mediated Fusion

The TMDs of viral fusion proteins generally encode a higher proportionof glycine residues relative to non-fusion proteins. Within the FASTprotein family, conflicting results have been found regarding the roleof transmembrane glycine residues. The p14 TMD seems to have no primarysequence requirements, whereas the N-terminal half of the ARV p10 TMDcontains a triglycine motif that is essential for fusion. The p15 TMD onthe other hand, has two glycine residues and a cluster of three serinesin the N-terminal half of its TMD. To determine the role, if any, ofthese p15 TMD motifs, the glycine residues were individually substitutedwith alanine residues to create p15G27A and p15G33A, and substitutionsof the tri-serine motif were also made in which the three serineresidues were cumulatively substituted with alanine residues (FIG. 14A).Each construct was transfected into QM5 cells, and the relative fusioncapabilities determined by syncytial indexing. Both glycinesubstitutions had a dramatic effect on membrane fusion; p15G27A producedno visible syncytia at 10 hours post-transfection while thesyncytiogenic activity of p15G33A was reduced by ˜75% (FIG. 14B).Similarly, replacement of even one serine residue (p15S23A) produced adramatic decrease in fusion activity and each accumulated substitution(p15S23/24A and p15S23-25A) resulted in a corresponding further decreaseand eventual elimination of syncytium formation (FIG. 14B). As discussedabove for the N-terminal substitutions, the extent of pore formationmirrored syncytiogenesis; p15G33A 29 induced delayed pore formation thatexceeded that induced by p15G27A or p15S23A while multiple substitutionsof the tri-serine motif eliminated pore formation (data not shown). Boththe transmembrane glycine residues and the tri-serine motif aretherefore involved in p15-mediated syncytiogenesis.

Example 9: Construction of the p14-Bombesin Fusion Protein

The p14-Bombesin construct was cloned into the pFastbac vector betweenBamHI and XhoI (FIG. 15A). For the purposes of being able to determinethe utility of the construct to bind to its target, the p14 constructincluded two tags, namely an enterokinase (EK) cleavage site and 6histidine residues (His). In practice, the construct need not containsuch tags. PCR analysis of the p14-Bombesin construct digested withBamHI and XhoI confirmed that thep14-Bombesin fragment of 465 bp wasincorporated into the pFastbac vector (4.8 kbp). A baculovirusexpression system was used to produce the recombinant virus in DH10a BacE. coli cells. Expression of the recombinant virus in sf21 cells andverified protein production by Western blot analysis using a polyclonalrabbit p14 primary antibody was confirmed (FIG. 15B). Analysis of theconstruct confirmed the sequence of the p14-Bombesin fusion polypeptide(FIG. 16).

Example 10: Fusion Activity Assay Using Transfection of RecombinantPlasmid DNA

HT1080 td tomato cells were transfected with pFastbac plasmidscontaining either empty vector, p14, or p14-bombesin using LipofectamineLTX+Plus (FIG. 17A and FIG. 17B). As shown by cells stained red in FIG.17A, the p14-bombesin construct was taken up and expressed in the HT1080td tomato cells. Cells were also stained with SYTOX green nuclear stainand then the average number of nuclei per cell was measured usingfluorescence intensity (FIG. 17A). The level of syncytia formationindicates that the p14-bombesin construct was as efficient as p14 alonein causing the formation of syncytia (FIG. 17B).

The functionality of the p14-bombesin construct was further confirmed byvisualization of syncytia formation in QM5 quail muscle cells (FIG. 18).QM5 quail muscle cells were incubated with purified protein (p14 andp14-bombesin). Syncytia formation can be seen by the formation ofmultinucleate cells, which are caused by functional p14 proteins.

Example 11: p14-Bombesin Containing Liposomes Target PC-3 Prostate Cells

PC-3 prostate cancer cells have been previously shown to express thegastrin-releasing peptide receptor (GRP-R). As such, this cell line wasused to determine whether the p14-bombesin liposomes could selectivelytarget these cells and delivery their cargo of FITC-dextran (FIG. 19).Liposomes were formulated with no fusion protein, native p14, andp14-bombesin. As shown in the first column of FIG. 19, the p14-bombesincontaining liposomes were capable of delivering the FITC-dextran payloadto the PC-3 cells.

Flow cytometry confirmed that indeed the p14-bombesin liposomes werecapable of delivering a FITC-dextran payload to PC-3 prostate cells(FIG. 20). Moreover, the amount of FITC-dextran delivered by thep14-bombesin liposomes was more than that of liposomes containing onlyp14 or liposomes containing no fusogenic proteins.

Example 12: Preparation of Liposomes

Liposomes were prepared following the thin film method of liposomepreparation as described by Fenske and Cullis (supra). Briefly, stocklipids based on Table 1 were added to a glass flask with at least threeglass beads in the concentrations shown in Table 2. The glass flaskcontaining the lipids in chloroform was attach to a rotary evaporator.The flask was then immersed into attached water bath, preheated to ˜38°C. The flask was then rotated at a constant speed. The chloroform wasevaporated by vacuum for 1-2 hrs. A predetermined amount of LiposomeBuffer (Lip B, 10 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mM NaCl(sodium chloride), pH 7.4) was added to the flask to generatemultilamellar liposomes. The flask was flushed with N₂, shaken, andre-flushed with N₂. The flask was sealed for 1 hour at room temperature(22-25° C.). This procedure produced a stock of 20 mM multilamellarlipid vesicles.

TABLE 1 Chemical Molecular Chemical Abbreviation Structure Formula NameDOPC 18:1 (Δ9-Cis) C₄₄H₈₄NO₈P 1,2-dioleoyl-sn-glycero- 3-phosphocholineDOPE 18:1 (Δ9-Cis) C₄₁H₇₈NO₈P 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine PE PEG2000 16:0 PEG2000 C₁₂₉H₂₅₉N₂O₅₅P1,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] DOTAP 18:01 C₄₂H₈₀NO₄Cl1,2-dioleoyl-3- trimethylammonium-propane Cholesterol cholesterolC₂₇H₄₆O cholesterol DC-Cholesterol cholesterol C₃₂H₅₇N₂O₂Cl3β-[N-(N′,N′- derivative dimethylaminoethane)- carbamoyl] cholesterol

TABLE 2 Desired molar ratio (%) Extreme Inner Outer Standard StrongExtreme Cationic Wrapo- Wrapo Lipids Profile Cationic Cationic II somesome DOPC 60 30 0 0 0 0 DOPE 30 30 0 25 0 0 Cholesterol 4 8 0 0 0 0PE-PEG2000 0 0 0 0 0 0.5 DOTAP 0 30 100 75 100 99.5 DC- 6 2 0 0 0 0Cholesterol Total %: 100 100 100 100 100 100

Unilamellar liposomes were produced using the LiposoFast-Basic unit(AVESTIN, Inc., 2450 Don Reid Dr. Ottawa, ON, CANADA K1H 1E1). This unitproduces unilamellar liposomes by the manual extrusion of amultilamellar liposome suspension through a polycarbonate membrane ofdefined pore size, using gas-tight, glass syringes. The sample is passedthrough the membrane by pushing the sample back and forth between twosyringes. 50 nm unilamellar liposomes were generated by extrusion using400 nm polycarbon filter (×20 movements through the membrane filter),and similarly with a 200 nm, 100 nm and 50 nm polycarbon filter untilthe desired size is achieved.

Liposomes were wrapped following the procedure described by Yagi et al.,(supra). Briefly, a wraposome core was created using 100% DOTAP andreconstitution with liposome buffer (as described above). The liposomecore was extruded to 50 nm as described above. The liposome core wasmixed with siRNA in a mole ratio of 1:26 siRNA:lipid. The differenttypes of siRNA used in the experiments are shown in Table 3. Thewraposome was dissolved with 100% ethanol. Since lipids will not formliposomes in ethanol, but instead remain in solution, the amount ofextra liposome buffer to be added was calculated to ensure that thefinal ethanol percentage is less than the maximum (i.e. the percentageof ethanol that will destabilize and dissolve the liposomal structures)to determine final volume required to attain the desired concentrationof wrapsomes. Liposomal buffer was added to the siRNA+core, followed byenvelope solution with constant, but moderate, vortexing. Ethanol wasthen removed from the liposomes.

Example 13: Liposomes were Shown to Contain siRNA

Relative siRNA concentrations were detected using gel electrophoresismethods. Serial dilutions of siRNA were electrophoresed and the signalof each sample determined by estimating the ethidium bromide signalbound to the siRNA (see FIG. 21). A standard curve was then generated.Alternatively, a known amount of siRNA was loaded and used as acomparison to quantitate siRNA loading. The signal associated with thesamples were then compared to the standard curve. The relative loadingefficiencies are shown in Table 4.

TABLE 3 Oligo Sense or Oligo 5′- Sequence Set # Antisense Name Label(5′--->3′) 1 Sense GFP_s — GGCUACGUCCAGGAG CGCACC   (SEQ ID NO: 20) 1Antisense GFP_as — GCGCUCCUGGACGUA GCCUU (SEQ ID NO: 21) 2 Sense ALCAM_s— AAGCCCGAUGGCUCC CCAGUAUU   (SEQ ID NO: 22) 2 Antisense ALCAM_as —AAUACUGGGGAGCCA UCGGGCUU   (SEQ ID NO: 23) 3 Sense SSB_s —ACAACAGACUUUAAU GUAAUU   (SEQ ID NO: 24) 3 Antisense SSB_as —UUACAUUAAAGUCUG UUGUUU   (SEQ ID NO: 25) 4 Sense Control_s —UCUUUUAACUCUCUU CAGGTT   (SEQ ID NO: 26) 4 Antisense Control_as —CCUGAAGAGAGUUAA AAGAU   (SEQ ID NO: 27) 5 Sense ALCAM_s Cy5AAGCCCGAUGGCUCC CCAGUAUU   (SEQ ID NO: 28) 5 Antisense ALCAM_as Cy5AAUACUGGGGAGCCA UCGGGCUU  (SEQ ID NO: 29)

TABLE 4 Liposomes siRNA % Loading +p14 ALCAM 34.5 −p14 ALCAM 40 +p14Control 33.3 −p14 Control 30.2

Example 14: Preparation of FAST Protein Containing Liposomes

For any liposomal preparation especially where a lipid profile isaltered in any way, an optimal n-octyl β-D-glucopyranoside (OG)concentration for inserting FAST protein should be determined. Theoptimal concentration is just below the critical OG concentration whichcauses dissolution of the liposomes.

Insertion of the p14 FAST protein into the membrane of the liposome wasaccomplished generally by the detergent depletion method. Briefly, thepurified FAST protein was reconstituted into liposomes by mixingdetergent-suspended FAST protein with liposomes pre-saturated withdetergent, followed by removal of the detergent.

Example 15: General Procedure for Studying siRNA Function

HT1080 cells were seeded on cover slips in 24 or 12 well cell cultureplates and grown overnight. Seeding density was such that 50-60%confluence was achieved. The ability to see single cells is desired.Cells were gradually cooled to 4° C. by placing the plates in a standardrefrigerator. Cells were washed gently with similarly cooled PBS.Liposomes with various cargoes were added to a final maximum of 1 mMlipid and incubated at 4° C. for 60 min. Cells were gently washed withcold PBS to then remove any unbound liposomes. Pre-warmed (37° C.) PBSwas then added to initiate delivery of liposomal cargo. At theappropriate time, cells were rapidly cooled to 4° C. in ice water andthen fixed (15 min in 1% formalin) and mounted with DAPI/Prolong Gold(Invitrogen). Slides were sealed to maintain the fluorescent signal foras long as possible. Fluorescent signal detection, imaging andquantitation was performed using an upright epifluorescent microscopewith a motorized Z stage (Zeiss AxioImager.Z1 microscope, Carl Zeiss,Thornwood, N.Y., USA) controlled by Volocity (Version 5.4, Improvision,Lexington, Mass.). All exposures were kept constant across sampleswithin the same experiment, and all contrasting for image presentationwas similarly kept constant for all fluorescent channels. A minimum of 5fields of view were analyzed and at least eighty individual cells areimaged and data quantitated per field of view. Graphing and statisticswere performed using GraphPad Prism (version 4.00 for Windows, GraphPadSoftware, San Diego Calif., USA).

Example 16: Uptake of siRNA into Cancer Cells was Enhanced by FASTProtein Containing Liposomes

Liposomes containing DOPC:DOPE:Cholesterol:DC-Cholesterol:PC-NBD:PC in aratio of 58:30:4:6:1:1 were prepared as described above and siRNAintroduced in a 1:26 siRNA:lipid mole ratio. P14 was added to themembrane of the liposome as described above.

FIG. 22A shows that FAST protein containing liposomes are capable ofdelivering siRNA more rapidly to cells than standard liposomes, whichdid not contain the FAST protein in their membranes. Twenty-four hoursafter incubation, the FAST protein containing liposomes increased theamount of siRNA in the cells compared to standard liposomes (FIG. 22B).

Example 17: FAST Protein Containing Liposomes Enhance siRNA-MediatedKnockdown of Functional Protein Compared to Standard Liposomes

The expression of GFP in HT-1080 cells was assessed using fluorescentquantitation. Cells that had undergone no treatment were used asbaseline estimation (100% expression). All other treatments werenormalized to this expression level (relative signal intensity/cell). Inthese experiments, FAST protein containing liposomes were able togenerate a significant reduction in GFP expression when compared to themedia controls, and the standard liposomes (see FIG. 23A and FIG. 23B).No statistical significant difference was noted between thelipofectamine treatments and the FAST protein containing liposometreatments. However, the FAST protein containing liposomes were moreeffective in reducing GFP expression than lipofectamine treated cells.

What is claimed is:
 1. A recombinant polypeptide for facilitating membrane fusion, said recombinant polypeptide comprising: an ectodomain sequence having the sequence of SEQ ID NO:2 that comprises a functional myristoylation motif; a transmembrane domain comprising 23 amino acid residues, at least two hydrophobic, β-branched residues adjacent the ectodomain, three consecutive serine residues immediately adjacent the at least two hydrophobic, β-branched residues, and a glycine residue at positions 7 and 13 from the junction between the ectodomain and the first hydrophobic, β-branched residue; and an endodomain sequence having the sequence of SEQ ID NO:3 or SEQ ID NO:4.
 2. The recombinant polypeptide of claim 1, wherein the at least two hydrophobic, β-branched residues are selected from isoleucine and valine.
 3. The recombinant polypeptide of claim 1, wherein the transmembrane domain comprises a sequence having the sequence of SEQ ID NO:11.
 4. The recombinant polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:1.
 5. The recombinant polypeptide of claim 1, comprising 23 amino acid residues, at least two N-terminal hydrophobic, β-branched residues, three consecutive serine residues immediately adjacent the at least two hydrophobic, β-branched residues, and a glycine residue at positions 7 and 13 from the N-terminus of the polypeptide.
 6. The recombinant polypeptide of claim 9, wherein the at least two hydrophobic, β-branched residues are selected from isoleucine and valine.
 7. The recombinant polypeptide of claim 1, characterized as a fusion-associated small transmembrane protein.
 8. The recombinant polypeptide of claim 7, wherein the fusion-associated small transmembrane protein is selected from the family Reoviridae.
 9. The recombinant polypeptide of claim 7, wherein the fusion-associated small transmembrane protein is selected from the genus Orthoreovirus and Aquareovirus.
 10. The recombinant polypeptide of claim 9, wherein the genus Orthoreovirus comprises avian, mammalian and reptilian reoviruses.
 11. The recombinant polypeptide of claim 7, wherein the fusion-associated small transmembrane protein is a p10 protein, a p14 protein, a p15 protein, or a p22 protein, or a chimera of two or more domains thereof.
 12. The recombinant polypeptide of claim 7, wherein the fusion-associated small transmembrane protein is operably linked to a targeting ligand.
 13. The recombinant polypeptide of claim 1, wherein the targeting ligand is bombesin.
 14. The recombinant polypeptide of claim 1, comprised within a liposome.
 15. An isolated nucleic acid molecule encoding the recombinant polypeptide of claim
 1. 16. The isolated nucleic acid molecule of claim 15, that comprises the sequence of SEQ ID NO:5.
 17. An expression vector comprising the isolated nucleic acid molecule of claim
 15. 18. A recombinant polypeptide for facilitating membrane fusion, said recombinant polypeptide comprising: an ectodomain sequence from a p14 fusion-associated small transmembrane (FAST) protein; a transmembrane domain comprising 23 amino acid residues, at least two hydrophobic, β-branched residues adjacent the ectodomain, three consecutive serine residues immediately adjacent the at least two hydrophobic, β-branched residues, and a glycine residue at positions 7 and 13 from the junction between the ectodomain and the first hydrophobic, β-branched residue; and an endodomain sequence from a p15 FAST protein.
 19. A composition comprising: (a) the recombinant polypeptide of claim 1 or claim 18; and (b) a diluent, a liposome, a buffer, or any combination thereof.
 20. A nucleic acid molecule encoding the recombinant polypeptide of claim 1 or claim
 18. 21. An isolated host cell comprising an expression vector that encodes the recombinant polypeptide of claim 1 or claim
 18. 22. A recombinant host cell comprising an expression vector that encodes a recombinant polypeptide that comprises: (1) an ectodomain sequence having the sequence of SEQ ID NO:2 that comprises a functional myristoylation motif; (2) a transmembrane domain comprising 23 amino acid residues, at least two hydrophobic, β-branched residues adjacent the ectodomain, three consecutive serine residues immediately adjacent the at least two hydrophobic, β-branched residues, and a glycine residue at positions 7 and 13 from the junction between the ectodomain and the first hydrophobic, β-branched residue; and (3) an endodomain sequence having the sequence of SEQ ID NO:3 or SEQ ID NO:4.
 23. A kit comprising, in a suitable container, (a) the composition of claim 19 or the recombinant host cell of claim 22; and (b) instructions for using the recombinant polypeptide in a method of inducing cell-cell fusion and heterokaryon formation in an animal cell. 